Background: Testicular cancer (TC) is the most common cancer diagnosed in men of reproductive age group. Sperm banking is recommended in these patients prior to cancer treatment. There is no literature describing the proteins dysregulated in the spermatozoa of TC patients with poor motility. Objective: The primary objective of this study was to compare the differences in the sperm proteome of normozoospermic (motility > 40%) and asthenozoospermic (motility < 40%) TC patients who had cryopreserved semen samples before initiating cancer therapy. Materials and methods: Pooled sperm samples from healthy fertile men (n = 8), normozoospermic (n = 20), and asthenozoospermic (n = 11) TC patients were used for quantitative global proteomic profiling by liquid chromatography-tandem mass spectrometry (LC-MS). The functional bioinformatic analysis was done by ingenuity pathway analysis software. Key differentially expressed proteins (DEPs) associated with binding of zona pellucida (CCT3), mitochondrial dysfunction (ATP5A1 and UQCRC2), sperm motility (ATP1A4), and an exosomal protein involved in the metabolic process of the spermatozoa (MMP9) were validated using Western blot analysis by comparing normozoospermic (n = 10) with asthenozoospermic (n = 10) TC patients. Statistical analysis was conducted using Mann-Whitney test. Results: A total of 813 and 957 proteins were detected in spermatozoa of normozoospermic and asthenozoospermic TC patients, respectively. On the other hand, 1139 proteins were detected in the spermatozoa of healthy fertile men. 198 proteins were identified as DEPs between TC patients with normal and abnormal semen parameters. Validation of DEPs revealed downregulation of key proteins (CCT3, ATP1A4, ATP5A1, and UQCRC2) implicated in the reproductive function in asthenozoospermic TC patients. Discussion: DEPs involved in the reproductive function pathways suggest defective spermatogenesis and maturation process in asthenozoospermic TC patients. Furthermore, dysfunctional exosomal pathway may be a possible cause of infertility in TC patients. Conclusion: The proteins associated with sperm function and fertilization process are compromised in TC patients irrespective of their semen parameters.