Nicotine, the main toxic substance in cigarette smoke, significantly reduced the differentiation and maturation ratio of Leydig cell in murine testes. To investigate the underlying mechanism, C57BL/6J mice were divided into control (CT) and nicotine treated (NT) groups. Next generation RNA sequencing and bio-informatics analysis were carried out to analysis the effects of nicotine on the RNA profile of Leydig cells. Expression level of 7 pathways remarkably changed after nicotine treatment. As the positive regulating pathway of Leydig cell differentiation, Hedgehog signaling pathway was found among these pathways. PTCH1 and beta-TrCP were down-regulated in nicotine treated mice Leydig cells, while GSK3 beta, Gli2 and Gli2 fragments increased significantly. Nicotine stimulated the destabilization of Gli2 via beta-TrCP induced ubiquitination and degradation. Gli2 was phosphorylated by up-expressed GSK3 beta during this process. Destabilization of Gli2 reduced the activation rate of target genes of Hedgehog signaling pathway such as Ptch1. The differentiation of Leydig cell positively regulated by Hh pathway was thus inhibited by nicotine exposure. Consequently, the male reproduction process powered by Leydic cell mediated androgen secretion was thus influenced. In conclusion, we find that nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway. (C) 2019 Elsevier Inc. All rights reserved.