Obesity is a metabolic disease and its relation with male subfertility has aroused a growing concern. However, it is unclear whether gene expression and post-translational modifications (PTMs), two vital molecular mechanisms regulating cellular functions, are associated with obesity-induced male reproductive dysfunction. In this study, male obesity with compromised sperm motility was induced by a high-fat diet (HFD) using a mouse model. The expression of motility related-genes, the level of histone modifications, and the global profiles of post-translational modifications (PTMs), were examined in testes of HFD and control mice by quantitative real-time PCR and western blot, respectively. Outer dense fiber protein 2, a major component of outer dense fibers in the sperm tail, is the most obviously down-regulated gene out of 11 evaluated genes, showing a reduction of about 50% RNA level in testes of obese male mice compared with that in control mice. Semi-quantitative analysis of the western blot demonstrated that ∼56% enrichment of di-methylated histone (H)3 lysine (K)36, ∼59% enrichment of 2-hydroxyisobutyrylated H4K8, ∼32% decrease of propionylated H3K23, ∼33% decrease of crotonylated H4K8, and ∼45% decrease of acetylated H3K122 and H4K8 were detected in testes of male HFD mice compared with that in control mice. In addition, male obesity up-regulated the testicular levels of ubiquitination by ∼18%, tyrosine nitration by ∼20%, lysine succinylation by ∼25%, lysine benzoylation by ∼28%, lysine malonylation by ∼32%, lysine glutarylation by ∼36%, lysine propionylation by ∼42%, lysine 2-hydroxyisobutyrylation by ∼45%, and SUMO1 modification by ∼59%, and down-regulated the testicular levels of O-GlcNAcylation by ∼12%, lysine crotonylation by ∼22%, and lysine acetylation by 35%. These findings indicate that altered gene expression and PTMs are associated with the obesity-induced male reproductive dysfunction.