Abstract Most studies in type 1 diabetes (T1D) have focused on the loss of the pancreatic beta-cell population. However, despite the involvement of the alpha-cell in the aetiology and complications of T1D, little is known about the regulation of the pancreatic alpha-cell mass in this disease. The need for a better understanding of this process is further emphasized by recent findings suggesting that alpha-cells may constitute a potential reservoir for beta-cell regeneration. In this study, we characterized the pancreatic alpha-cell mass and its regulatory processes in the transgenic RIP-B7.1 mice model of experimental autoimmune diabetes (EAD). Diabetic mice presented insulitis, hyperglycaemia, hypoinsulinemia and hyperglucagonemia along with lower pancreatic insulin content. While alpha-cell mass and pancreatic glucagon content were preserved at the early-onset of EAD, both parameters were reduced in the advanced phase. At both stages, alpha-cell size, proliferation and ductal neogenesis were up-regulated, whereas apoptosis was almost negligible. Interestingly, we found an increase in the proportion of glucagon-containing cells positive for insulin or the beta-cell transcription factor PDX1. Our findings suggest that pancreatic alpha-cell renewal mechanisms are boosted during the natural course of EAD, possibly as an attempt to maintain the alpha-cell population and/or to increase beta-cell regeneration via alpha-cell transdifferentiation.