Sperm motility is a prerequisite for achieving pregnancy, and alterations in sperm motility, along with sperm count and morphology, are commonly observed in subfertile men. The aim of the study was to determine whether the expression level of genes annotated with the Gene Ontology (GO) term ‘sperm motility’ differed in sperm collected from healthy men and men diagnosed with oligoasthenozoospermia. Reverse transcription quantitative real-time PCR (RT-qPCR), quantitative mass spectrometry (LC-MS/MS), and enrichment analyses were used to validate a set of 132 genes in 198 men present at an infertility clinic. Out of the 132 studied sperm-motility-associated genes, 114 showed differentially expressed levels in oligoasthenozoospermic men compared to those of normozoospermic controls using an RT-qPCR analysis. Of these, 94 genes showed a significantly lower expression level, and 20 genes showed a significantly higher expression level. An MS analysis of sperm from an independent cohort of healthy and subfertile men identified 692 differentially expressed proteins, of which 512 were significantly lower and 180 were significantly higher in oligoasthenozoospermic men compared to those of the normozoospermic controls. Of the 58 gene products quantified with both techniques, 48 (82.75%) showed concordant regulation. Besides the sperm-motility-associated proteins, the unbiased proteomics approach uncovered several novel proteins whose expression levels were specifically altered in abnormal sperm samples. Among these deregulated proteins, there was a clear overrepresentation of annotation terms related to sperm integrity, the cytoskeleton, and energy-related metabolism, as well as human phenotypes related to spermatogenesis and sperm-related abnormalities. These findings suggest that many of these proteins may serve as diagnostic markers of male infertility. Our study reveals an extended number of sperm-motility-associated genes with altered expression levels in the sperm of men with oligoasthenozoospermia. These genes and/or proteins can be used in the future for better assessments of male factor infertility.