The regulators of stem Leydig cell (LC) development remain largely unknown. The effect of glucagon-like peptide-1 (GLP-1) on rat stem LC proliferation and differentiation was investigated using a 3D tissue culture system and an ethane dimethane sulfonate (EDS)-treated in vivo LC regeneration model. RNA-seq analysis was performed to analyze pathways in which GLP-1 may be involved. GLP-1 (3 and 30 nmol/L) significantly increased medium testosterone levels and up-regulated the expression of Scarb1, Cyp11a1, and Hsd11b1. GLP-1 in vitro did not affect stem LC proliferation by EdU incorporation assay. Intratesticular injection of GLP-1 (10 and 100 ng/testis) into the LC-depleted testis from day 14 to day 28 post-EDS significantly increased serum testosterone levels and upregulated the expression of Cyp11a1, Hsd3b1, and Hsd11b1. It did not affect the number of HSD11B1+ and CYP11A1+ LCs. RNA-seq analysis revealed that GLP-1 upregulated several pathways, including cAMP-PKA-EPAC1 and MEK/ERK1/2. GLP-1 stimulates stem LC differentiation without affecting its proliferation, showing its novel action and mechanism on rat stem LC development. In brief: Glucagon-like peptide-1 stimulates stem Leydig cell development. Glucagon-like peptide-1 stimulates stem Leydig cell differentiation without affecting its proliferation.