We previously identified the Rab1A small GTPase as a conserved activator of mTORC1 in response to amino acid (AA) stimulation in yeast and mammalian cells. To better understand the physiological function of Rab1A- mTORC1 signaling in vivo, we genetically engineered conditional Rab1A knockout (Rab1A-KO) in mice. We found that tamoxifen-induced Rab1A knockout in young adult mice impairs insulin expression and beta cell proliferation, leading to hyperglycemia and glucose intolerance. Using beta-cell lines, we further found that Rab1A mediates AA signaling to regulate insulin transcription and beta cell proliferation through phosphorylation of the transcription factor Pdx1. Our results revealed a novel role of AA-Rab1A-mTORC1 signaling in controlling whole body’s glucose homeostasis in mammals. The objective of this study is to use human patient samples to obtain clinical evidence that Rab1A plays an important role in the progression of diabetes mellitus.