Objective To explore the effect of microRNA (miRNA)-301a-3p on chemotherapy sensitivity and its possible mechanism in human colorectal cancer cells HCT-8.Methods The chemotherapy sensitivity of HCT-8 and HCT-8/FU cells was determined by CCK-8，and the expression of miRNA301a3p and PTEN in the HCT-8 and HCT-8/FU cells was measured by RTPCR. Transfection of miRNA-301a-3p inhibitor was used to downregulate the miRNA-301a-3p levels in HCT-8/FU cells. The chemotherapy sensitivity was measured by CCK-8. Bioinformatics prediction software was used to miRNA-301a-3p target gene predictive analysis and paris of luciferasw reporter system verification miRNA-301a-3p target gene. Effect of PTEN expressioninhibited on the chemotherapy sensitivity of HCT-8/FU cell line.The influence of suppression of miRNA-301a-3p expression on the expression of PTEN,AKT and pAKT were observed.Results The chemotherapy sensitivity of HCT-8/FU cell line was lower than that of HCT8 cell line. Expression levels of miRNA301a3p in HCT-8/FU cell line was higher than that of HCT-8 cell line. The expression levels of PTEN in HCT-8/FU cell line was lower than that in HCT-8 cell line. MiRNA-301a-3p inhibitor reduced HCT-8/FU cell proliferation and enhanced chemotherapy sensitivity. PTEN was identified and validated to be a target gene of miRNA-301a-3p. Inhibition of the PTEN gene can reduce the chemotherapy sensitivity and inhibition of the miRNA-301a-3p could increase PTEN expression and reduce pAKT expression.Conclusion Downregulation of miRNA-301a-3p increases the chemotherapy sensitivity of HCT8/FU cell line. The mechanism was at least partly due to miRNA301a3p/PTEN/AKT signaling pathway.