Objective To explore the effects of matrine on gemcitabine (GEM) resistance in pancreatic cancer and to provide a new therapeutic strategy for restoring the chemosensitivity of pancreatic cancer cells. Methods The pancreatic cancer cell line AsPC-1 was selected and GEM resistance induced by treatment with gemcitabine. The drug- resistant cell line AsPC-1-GEM was established and confirmed by CCK-8. The effect of matrine on AsPC-1-GEM was analyzed by detecting the cell viability. Expression changes to multidrug resistance protein 2 (ABCG2) and the effect of matrine on ABCG2 were further tested using RT-PCR and Western Blot. We further established a GEM-resistant cell line xenograft model of pancreatic cancer in nude mice and observed the changes in tumor volume after treatment with matrine. The effect of matrine on the expression of ABCG2 was detected by immunohistochemistry. Results ABCG2 expression was increased in GEM-resistant AsPC-1 cells (P< 0. 01), and matrine significantly reduced ABCG2 expression (P< 0. 01) and improved the sensitivity of AsPC-1-GEM cells (P< 0. 01). Treatment with matrine reduced the expression of ABCG2 (P< 0. 01) and retarded tumor growth ( P< 0. 01) in the xenograft models with AsPC-1-GEM tumors. Conclusions Matrine increases the sensitivity of GEM-resistant pancreatic cancer cell lines by decreasing the expression of ABCG2.