The aim of the present study was to identify microRNAs (miRNAs) that regulate the proliferation and differentiation of Leydig cells (LCs) of rat. Three small RNA libraries derived from progenitor LCs (PLCs), immature LCs (ILCs) and adult LCs (ALCs) were analyzed by microarrays. In total, 68 differentially expressed miRNAs (DEMs) were identified. Based on the trend of DEM expression from PLCs to ALCs, primary LCs were transfected with miRNA mimics or inhibitors. Five miRNAs (miR-30a-5p, miR-3585-5p, miR-212-3p, miR-369-5p and miR-434-3p) promoted PLC proliferation, and 3 miRNAs (miR-17-5p, miR-532-3p and miR-329-3p) activated caspase-3, which triggered LC apoptosis. For steroidogenesis, 18 miRNAs could elevate or inhibit androsterone release at the PLC stage. Eleven and 9 miRNAs inhibited the production of 5α-androstane-3α,17β-diol in ILCs and testosterone in ALCs, respectively. miR-17-5p, miR-29a-3p and miR-299a-5p decreased androgen production by LCs at all developmental stages. Furthermore, the miR-299a-5p-mediated decrease in androgen production by the LC lineage was primarily achieved by downregulating the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). These findings provide insights into the regulatory roles of miRNAs during the postnatal development of LCs and suggest potential strategies for the treatment of steroid-related disorders.