SummaryObesity, an increasingly frequent societal disease can also be accompanied by declines in spermatozoa quality and male subfecundity. To determine if there are obesity‐associated proteomic changes potentially affecting sperm quality and motility, differential proteomic analysis was performed on spermatozoa from both obesity‐associated asthenozoospermia and clinically healthy individuals, using a label‐free quantitative LC‐MS/MS approach. We resolved 1975 proteins in the human sperm proteome, amongst which, 105 proteins were less abundant, whereas 22 other proteins increased in obesity‐associated asthenozoospermia. Functional category analyses indicated that the differentially expressed proteins are mainly related to cytoskeletal regulation, vesicle biogenesis, metabolism, and protein degradation involved in spermiogenesis and sperm motility. Furthermore, declines in endoplasmic reticulum protein 57 (ERp57) and actin‐binding‐related protein T2 (ACTRT2) expression were verified by immunofluorescence, Western blot, and flow cytometry analyses. It is evident that ERp57 is localized in the acrosome region, neck and principal piece of human spermatozoa, whereas ACTRT2 is localized in the post‐acrosomal region and middle piece. Thus, these differences in protein expression in asthenozoospermia may contribute to the underlying sperm quality defects afflicting these individuals. Notably, declines in ERp57 and ACTRT2 expression in obesity‐associated asthenozoospermia may play critical roles in reducing sperm motility.