Abstract：Objective：To investigate the effects of different concentrations of paeonol on proliferation， migration and invasion of bladder cancer 5637 cells in vitro and the regulation on cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). Methods：The effect of different concentrations of paeonol on the proliferation of bladder cancer 5637 cells was investigated by observing the survival rate of cells treated with different concentrations of paeonol for 24，48 and 72 hours and by calculating the half inhibitory concentration (IC50). Cell scratch experiment and Transwell invasion experiment were used to observe the effect of different concentrations of paeonol on the migration and invasion of bladder cancer 5637 cells. Enzyme-Linked Immunosorbent Assay (ELISA) was used to determine the effects of paeonol on cyclooxygenase-2 (COX-2)， prostaglandin E2(PGE2) and COX-2 activity in bladder cancer 5637 cells treated with paeonol at different concentrations for 24 hours. Results： The survival rate of bladder cancer 5637 cells decreased gradually with the increase of paeonol concentration. At the 72 hours，200 μg/mL of paeonol had the most obvious inhibitory effect on cell proliferation. At the 24 hours，48 hours，72 hours，the respective cell survival rates of bladder cancer 5637 cells in the 25 μg/mL group， 50 μg/mL group， 100 μg/mL group and 200 μg/mL group were lower than those in the control group，differences being significant (P＜0.05). At the 24 hours， the cell survival rates in the 50 μg/mL group， 100 μg/mL group and 200 μg/mL group were respectively lower than those in the 6.25 μg/mL group，12.5 μg/mL group(P＜0.05)；the cell survival rates in the 100 μg/mL group，200 μg/mL group were respectively lower than those in the 25 μg/mL group，50 μg/mL group (P＜ 0.05)；the cell survival rate in the 200 μg/mL group was lower than that in the 100 μg/mL group (P＜0.05). At the 48 hours，72 hours，the cell survival rates in the 50 μg/mL group，100 μg/mL group and 200 μg/mL group were respectively lower than those in the 6.25 μg/mL group，12.5 μg/mL group and 25 μg/mL group (P＜0.05)； the cell survival rates in the 100 μg/mL group，200 μg/mL group were respectively lower than those in the 50 μg/mL group (P＜0.05)，and the cell survival rates in the 200 μg/mL group was lower than those in the 100 μg/mL group (P＜0.05). The IC50 of paeonol on 5637 cells at the 24 hours， 48 hours， and 72 hours were 88.27，74.24 and 77.07 μg/mL，respectively. The cell scratch experiment showed that the migration of 5637 cells of bladder cancer was inhibited after 24-hour treatment with paeonol at different concentrations (25，50，100，200 μg/mL)，which indicated a concentration-dependent manner，and the scratch healing rate in all groups was respectively lower than that in the control group (P＜0.05). The results of Transwell invasion experiment showed that the invasion of 5637 cells of bladder cancer was inhibited after treatment with paeonol at different concentrations (25， 50， 100， 200 μg/mL) for 24 hours. As the concentration increased，the number of cells passing through the microporous membrane decreased. The numbers of transmembrane cells in 25μg/mL group， 50 μg/mL group， 100 μg/mL group and 200 μg/mL group were respectively lower than that in the control group (P＜0.05). The numbers of transmembrane cells in the 50 μg/mL group，100 μg/mL group and 200 μg/mL groups were respectively lower than that in the 25 μg/mL group (P＜0.05)； the numbers of transmembrane cells in the 100 μg/mL group and 200 μg/mL group were respectively lower than that in the 50 μg/mL group (P＜0.05)；the number of transmembrane cells in the 200 μg/mL group was lower than that in the 100 μg/mL group (P＜0.05). The contents of COX-2 and PGE2 and the activity of COX-2 in the superserum of bladder cancer cell 5637 decreased with the increase of paeonol concentration ； the contents of COX-2 and PGE2 and the activity of COX-2 in the 25 μg/mL group，50 μg/mL group，100 μg/mL group and 200 μg/mL group were lower than those in the control group，differences being significant (P＜0.05). The PGE2 content in the 50 μg/mL group was lower than that in the 25 μg/mL group (P＜0.05)，and the contents of COX-2 and PGE2 and the activity of COX-2 in the 100μg/mL group and 200 μg/mL group were lower than those in the 25 μg/mL group (P＜0.05)；the PGE2 content in the 100 μg/mL group was lower than that in the 50 μg/mL group (P＜0.05)， and the contents of COX-2 and PGE2 and the activity of COX-2 in the 200 μg/mL group were lower than those in the 50 μg/mL group， 100 μg/mL group (P＜0.05). Conclusion： The mechanism by which paeonol can inhibit the proliferation，migration and invasion of bladder cancer 5637 cells may be related to the reduction of COX-2 content，PGE2 content and COX-2 activity.