Testosterone is primarily produced by Leydig cells of the mammalian male gonads. The cellular functions of Leydig cells are regulated by the hypothalamus–pituitary–gonad axis, whereas the microRNA (miRNA) changes of LH-treated Leydig cells are unknown. Mouse TM3 Leydig cells were treated with LH, and deep sequencing showed that 29 miRNAs were significantly different between two groups (fold change of >1.5 or <0.5, p < .05), of which 27 were upregulated and two were downregulated. The differential expression of miR-29b-3p, miR-378b, miR-193b and miR-3695 was confirmed by quantitative real-time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs were involved in the Wingless and INT-1, adenosine 5′-monophosphate-activated protein kinase, NF-kappa B and Toll-like receptor signalling pathways. Results showed that miRNAs might be involved in the regulation of LH to Leydig cells.