During pregnancy a high rate of beta-cell proliferation occurs, making of this a useful model for the study of islet cell expansion in vivo. We used the murine pregnancy model to assess the effect of Rapamycin treatment on islet cell proliferation in vivo. Rapamycin is routinely used for the prevention of graft rejection in transplanted patients, including islet transplant recipients. As expected, pregnancy led to increased beta-cell proliferation, islet yield and skewing in size distribution after isolation and pancreatic insulin content, when compared to non-pregnant females. Rapamycin treatment resulted in reduced beta cell proliferation in pregnant mice, while minimal effects of Rapamycin treatment were observed on islet function both in vivo and in vitro. Rapamycin treatment of islets resulted in reduced phosphorylation of p70s6k, a downstream effector molecule of mTOR and increased ERK1/2 phosphorylation. In conclusion, beta-cell replication is reduced under Rapamycin treatment in vivo, suggesting that this mechanism may be operational and impair beta-cell renewal in transplanted patients.