AIM: To study the effects of Roux-en-Y gastric bypass (RYGB) on the expression of pancreatic duodenal homeobox- 1 (PDX-1) and pancreatic β-cell regeneration/ neogenesis, and their possible mechanisms in diabetics. METHODS: Three groups of randomly selected nonobese diabetic Goto-Kakizaki (GK) rats were subjected to RYGB, sham-RYGB and sham-operation (sham-op) surgery, respectively. The rats were euthanized at postoperative 1, 2, 4 and 12 wk. Their pancreases were resected and analyzed using reverse transcription polymerase chain reaction to detect the mRNA of PDX-1. Anti-PDX-1 immunohistochemical (IHC) staining and Western blotting were used to detect the protein of PDX-1. Double IHC staining of anti-Brdu and -insulin was performed to detect regenerated β-cells. The index of double Brdu and insulin positive cells was calculated. RESULTS: In comparison with sham-RYGB and sham-op groups, a significant increase in the expressions of PDX-1 mRNA in RYGB group was observed at all experimental time points (1 wk: 0.378 ± 0.013 vs 0.120 ± 0.010, 0.100 ± 0.010, F = 727.717, P < 0.001; 2 wk: 0.318 ± 0.013 vs 0.110 ± 0.010, 0.143 ± 0.015, F = 301.509, P < 0.001; 4 wk: 0.172 ± 0.011 vs 0.107 ± 0.012, 0.090 ± 0.010, F = 64.297, P < 0.001; 12 wk: 0.140 ± 0.007 vs 0.120 ± 0.010, 0.097 ± 0.015, F = 16.392, P < 0.001); PDX-1 protein in RYGB group was also increased significantly (1 wk: 0.61 ± 0.01 vs 0.21 ± 0.01, 0.15 ± 0.01, F = 3031.127, P < 0.001; 2 wk: 0.55 ± 0.00 vs 0.15 ± 0.01, 0.17 ± 0.01, F = 3426.455, P < 0.001; 4 wk: 0.39 ± 0.01 vs 0.18 ± 0.01, 0.22 ± 0.01, F = 882.909, P < 0.001; 12 wk: 0.41 ± 0.01 vs 0.20 ± 0.01, 0.18 ± 0.01, F = 515.833, P < 0.001). PDX-1 mRNA and PDX-1 protein production showed no statistical significance between the two sham groups. Many PDX-1 positive cells could be found in the pancreatic islets of the rats in RYGB group at all time points. In addition, the percentage of Brdu-insulin double staining positive cells was higher in RYGB group than in the other two groups (1 wk: 0.22 ± 0.13 vs 0.03 ± 0.06, 0.03 ± 0.06, P < 0.05; 2 wk: 0.28 ± 0.08 vs 0.00 ± 0.00, 0.03 ± 0.06, P < 0.05; 4 wk: 0.24 ± 0.11 vs 0.07 ± 0.06, 0.00 ± 0.00, P < 0.001; 12 wk: 0.20 ± 0.07 vs 0.03 ± 0.06, 0.00 ± 0.00, P < 0.05). CONCLUSION: RYGB can increase the expression of pancreatic PDX-1 and induce the regeneration of β-cells in GK rats. The associated regeneration of islet cells may be a possible mechanism that how RYGB could improve type 2 diabetes mellitus.