Abstract The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment.