The aim of this study was to investigate the effects of limited food intake (LFI) (24, 48 and 120 h) and a single i.p. dose of vitamin C supplementation (500 mg/kg) on serum glucose and C-peptide levels, and pancreatic insulin and glucagon levels in guinea pigs. The highest serum glucose levels were found after vitamin C supplementation plus LFI for 48 h (LFI 48). Serum C-peptide levels were not significantly affected by food limitation (LFI 24, LFI 48, or LFI 120) as compared with controls, but when vitamin C was supplemented, the C-peptide levels were moderately enhanced. Immunohistochemical findings on pancreatic islets showed increased staining intensity for both insulin and glucagon when vitamin C was supplemented. In addition, the alpha and beta cells were stimulated, particularly by vitamin C supplementation plus LFI 120. Based on these findings, vitamin C supplementation may have a beneficial effect on the alpha and beta cells.