125I seed implantation has recently become an effective, safe, and feasible treatment for advanced pancreatic cancer in China. Gemcitabine (GEM), superior to fluorouracil, has been widely proved as effective chemotherapy for many solid tumors and become the standard treatment for locally advanced and metastatic pancreatic cancer. The study aimed to evaluate the combined effect of 125I and GEM on pancreatic carcinoma cells (PANC-1) cells and explore the underlying molecular basis.,PANC-1 cells were treated with 125I continuously at a low dose of radiation, combined with or without sensitizing concentration of GEM. The clonogenic capacity, cellular proliferation, cell cycle distribution, apoptosis, and molecular pathways of the cells following these treatments were analyzed in vitro.,The cell growth could be significantly inhibited after the treatment with GEM or 125I alone, while the inhibition effects would be greater with combination therapy than either monotherapy (72 h, C vs. GEM, t = 16.59, P < 0.01; C vs. 125I, t = 9.808, P < 0.05; C vs. 125I + GEM, t = 17.87, P < 0.01; 125I vs. 125I+GEM, t = 8.191, P < 0.05). GEM increased radiation-induced apoptosis (4 Gy, 125I vs. 125I+GEM, t = 10.43, P < 0.01) and induced the arrest of G1. Caspase-3 expression and the Bax/Bcl2 ratio were lower in cells receiving combination treatment than that of in cells treated with 125I or GEM alone.,The combined treatment of 125I and GEM-induced stronger anti-proliferation effect than single-treatment, due to the cell cycle arrest and more cellular apoptosis in PANC-1 cells. The increased Bax/Bcl-2 ratio may lead to enhanced apoptosis.