Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone predominantly taken as drugs of abuse. Using in vivo treatment of adult male rats we investigated the effects of testosterone enanthate (TE) a wide abused AAS, on apoptosis of Leydig cells. Increased testosterone and decreased luteinizing hormone levels in serum and decreased intra-testicular testosterone content were found in 2 and 10 weeks treated groups. Two weeks of TE-treatment decreased mitochondrial membrane potential and increased prevalence of Leydig cell apoptosis. The increased incidence of Leydig cell apoptosis returned to control levels after 10 weeks of TE-treatment but testes contained fewer Leydig cells. TE-treatment stimulated androgen receptor (AR) expression in Leydig cells which was followed with changed transcriptional pattern of genes related to mitogen activated protein kinase (MAPK) signaling. Two weeks of TE treatment increased expression of Mapk2k1 and Mapk11 (also known as p38 b). This was prevented by in vivo administration of androgen receptor blocker, suggesting Mapk11 involvement in AR associated increased apoptosis of Leydig cells. Additionally, results showed the decreased expression of extracellular signal-regulated kinase 1 (Erk1), Mapk7 and Mapk8 in both 2 and 10 weeks of TE-treatment but significantly increased the expression of Mapk2k2 in Leydig cells from 10 weeks treated rats. The expression of dual specificity protein phosphatase 1 (Dusp1) was decreased in 10 weeks of treatment while levels of Erk2 and Erk3 and p53 gene expression were not affected.