Abstract： Objective： To discuss the mechanism of Hedyotis Diffusae Herba- Scutellariae Barbatae Herba(HD-SB) extract in treating colorectal cancer based on transcriptome sequencing. Methods：Different concentrations(0， 0.25， 0.5， 1， 2， 4 mg/mL) of Hedyotis Diffusa Herba(HD) extract， Scutellariae Barbatae Herba(SB) extract and HD-SB extract were used for treating three colorectal cancer cell lines(HT- 29，HCT-116，SW620) for 24 hours，and the cell viability was detected by CCK8. The untreated SW620 cells(the control group) and SW620 cells which were treated with HD-SB at the concentration of 1 mg/ mL for 24 hours(the HD-SB group) were selected for transcriptome sequencing. The expression of five key genes， including winglesstype MMTV integration site family member 11(WNT11)， winglesstype MMTV integration site family member 2B(WNT2B)， frizzled homolog 2(FZD2)， low density lipoprotein receptorrelated protein 6(LRP6) and phospholipase C- β4(PLCβ4)， targeting winglesstype MMTV integration site family(WNT) pathway was detected by real- time PCR. Results：The HD，SB and HD- SB extracts at the concentration of 2.4 mg/mL can significantly inhibit cell viability of HCT-116，HT-29 and SW620(P＜0.05)； the viability of HT-29 and SW620 cells treated with HD-SB extract at the concentrations of 1 ， 2 and 4 mg/mL was significantly decreased when compared with that treated with HD extract and SB extract (P ＜ 0.05). Transcriptome sequencing showed that significant changes were found in the comparisons of 1 522 differential genes between the HD-SB group and the control group(P＜0.05)，of which 203 genes in the HD- SB group were increased(P＜0.05) and 1 319 genes were decreased(P＜0.05). GO functional enrichment analysis showed that the differential genes in mRNA were mainly related to functions of cell components， regulation of cell expression， and regulation of immune responses； KEGG signaling pathway enrichment analysis showed that differential genes were mainly involved in the metabolismrelated signaling pathways， cancer- related pathways， mitogen activated protein kinase(MAPK) signaling pathway， WNT signaling pathway and so on. The expressions of five key genes， including WNT11， WNT2B，FZD2，LRP6 and PLCβ4，of the WNT pathway in the HD-SB group were significantly decreased when compared with those in the control group(P＜0.05). Conclusion：The HD- SB extract can inhibit the proliferation of SW620 cells of colorectal cancer， and its mechanism may be related to the inhibition of WNT pathway activation.