Asthenozoospermia (AS), a common cause of male infertility, is characterized by reduced progressive sperm motility. To date, integrated proteomic comparisons of matched spermatozoa (S) and seminal plasma (SP) samples from AS patients remain largely unexplored. In this study, we performed data-independent acquisition (DIA)-based quantitative proteomic profiling of paired S and SP samples collected from 31 AS patients and 31 healthy controls (HCs).