To study the regulatory effect of Herba Hedyotis Diffusae on multidrug-resistance (MDR)-related microRNAs (miRNAs) in colorectal cancer，and further explore the underlying mechanisms of its MDR reversion.Methods:The signal pathway regulated by miRNA was forecasted by MTT assays，morphology observation，miRNA array analysis，QPCR validation，GO and Pathway analysis through treatment of HDW in HCT-8/5-FU cells.Results:HDW significantly inhibited colorectal cell viability and decreased density of HCT-8/5-FU cells.There were 23 up-regulated and 34 down-regulated in 57 miRNA differential expression after HDW treatment; and QPCR verification confirmed that HDW increased expression of miR-92b-5p，miR-1247-3p and miR-4800-5p and decreased expression of miR-4454，miR-4443，miR-483-5p; and the signal pathway prediction showed that HDW could control PI3K/Akt，TGF-β/Smad，MAPK，P53，Wnt and JAK-STAT signaling pathways regulating MDR，migration，invasion，proliferation and apoptosis.Conclusion:HDW can remarkably suppress proliferation in HCT-8/5-FU cells，and it is one of the mechanisms to reverse the drug resistance of colorectal cancer by regulating miRNAs expression.