Objective To investigate the expression of miR-101 in pancreatic cancer and the effect of down-regulation miR-101 on proliferation of pancreatic cancer cell line ASPC1. Methods Real-time PCR was used to determine the expression of miR-101 in pancreatic cancer, adjacent tissues and pancreatic cancer cell line ASPC-1. The miR-101 over-expression vector (peGFPc1-miR-101) was constructed and was transfected into ASPC-1 cell. Transfection efficiency was measured by fluorescence microscope. The expression of miR101in the transfected cells was detected by real-time PCR. Cell viability analysis was performed by MTT. The targeted genes of miR-101 in pancreatic cancer were scanned by the online targeted gene prediction software (target Scan). Results The expression of miR-101 was in pancreatic cancer tissues, adjacent tissues and ASPC-1 cell line, respectively. The expressions in pancreatic cancer tissues and ASPC-1 cells were significantly lower than that in adjacent tissues ( P ＜ 0.01 ). The expression of miR 101 in transfected cells increased to 19.8 folds as much as that in the control group (P ＜0.01 ). Proliferation rate of transfected cells was significantly decreased, which was only 26% of primary cells ( P ＜ 0.01 ). EZH2 was the potential targeted gene of miR-101 in pancreatic cancer. Conclusions miR-101 was weakly expressed and it may affect the proliferation of pancreatic cancer cell by inhibiting the EZH2 expression.