Objective：This study aims to observe the effects of disulfiram（DSF） on the proliferation and cell cycle arrest in human pancreatic cancer cells，and to explore its mechanism. Methods：CCK-8 was used to detect the effects of DSF on the proliferation activity and the cycle distribution was detected by flow cytometry. Real-time PCR and Western blot were used to detect mRNA and protein expression of GADD45A，G2/M related CCNB1，CDC25C，and CDK1 genes and proteins. The phosphorylation of P38 and JNK in MAPK pathway were also detected. SiRNA assay was used to detect the changes of cell cycle and phosphorylation of proteins in MAPK pathway after growth arrest and DNA damage inducible 45A（GADD45A） genes were decreased. Results：DSF combined with Cu（DSF/Cu）inhibited the proliferation of pancreatic cancer cells in concentration-dependent trend. GADD45A was significantly increased in DSF treated groups（11.4 times of control group in PANC-1 cells and 7.99 times of control group in PATU8988T cells，P < 0.001，respectively）. The cells were mainly blocked in G2/M phase and CCNB1，CDC25C and CDK1 genes were significantly decreased（31%，35%，37% of control in PANC-1 cells and 48%，24%，29% of control in PATU8988T cells，P < 0.05，respectively）after treated 24 hours. The proteins showed the same results with the mRNA expression while the p-P38 and p-JNK were increased in dosage and time-dependent trend. Compared with the DSF treated groups，the groups of cells pretreated with siRNA-GADD45A showed an increasing relative viability and the percentage of G2/M phase cells was decreased. Meanwhile，the p-P38 and p-JNK were also decreased. Conclusion：DSF/Cu can significantly inhibit the proliferation and induce G2/M arrest in pancreatic cancer cells. Its anti-tumor effect may attribute to upregulating GADD45A and activating JNK/P38 MAPK signaling pathway.