Arbutin, a widely used skin lightening agent, has raised concerns regarding its potential side effects. In this study, we investigated the impact of arbutin on Leydig cell function using an in vitro model. We measured medium androgen levels, as well as the gene and protein expression related to Leydig cell steroidogenesis. Rat immature Leydig cells from age of 35 days were exposed to arbutin at concentrations ranging from 0.5 to 50 μM for a duration of 3 hrs. Following treatment, we observed a significant inhibition of androgen secretion by Leydig cells at both the 5 and 50 μM concentrations of arbutin. Furthermore, at a concentration of 50 μM, arbutin effectively blocked the stimulatory effects of luteinizing hormone (LH) and 8Br-cAMP on androgen secretion. Subsequent analysis revealed that arbutin downregulated the expression of crucial genes involved in androgen production, including Lhcgr, Hsd3b1, Cyp17a1, and Srd5a1. In silico computer program analysis predicted that arbutin exhibits good absorption, possesses a long elimination half-life, and may have other potential toxicity such as hepatoxicity. Taken together, our results demonstrate that arbutin negatively influences Leydig cell function and androgen production, potentially impacting male reproductive health.