In attempt to elucidate molecular mechanisms and factors involved in beta cells regeneration, we evaluated a possible role of L-arginine-nitric oxide (NO)-producing pathway in alloxan-induced diabetes mellitus. Diabetes was induced in male Mill Hill rats with single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were additionally separated in three subgroups: one, receiving L-arginineHCl (2.25%), another L-NAMEHCl (0.01%) for 12 days as drinking liquids and the third was control. Treatment of diabetic animals started after diabetes induction (glucose level 12 mmol/l). We found that disturbed glucose homeostasis i.e. blood insulin and glucose levels in diabetic rats was restored after L-arginine treatment. Immunohistochemical findings revealed L-arginine favorable effects on beta cells neogenesis, i.e. it increased insulin immunopositive cells area. Moreover, confocal microscopy showed colocalization of insulin and pancreas duodenum homeobox-1 (PDX-1) in both endocrine and exocrine pancreas. This increase in insulin-expressing cells was accompained with increased cell proliferation (observed by proliferating cell nuclear antigen-PCNA immunopositivity) which occurred in regulated manner since it was associated by increased apoptosis (detected by TUNEL method). Furthermore, L-arginine enhanced both nuclear factor-kB (NF-kB) and neuronal nitric oxide synthase (nNOS) immunopositivities. The effect of L-arginine on antioxidative defense was observed especially in restoring to control level diabetes-induced increase in glutathione peroxidase activity. In contrast to L-arginine, diabetic pancreas was not affected by L-NAME supplementation. In conclusion, the results suggest beneficial L-arginine effects on alloxan-induced diabetes resulting from the stimulation of beta cell neogenesis including complex mechanisms of transcriptional and redox regulation.