Sperm motility is essential for fertilisation, yet despite this, there is a lack of information regarding how the motor complex of the flagella works. During ejaculation, spermatozoa transit from an immotile state, within the caudal regions of the epididymis, to a motile state once they reach the female reproductive tract. To gain further insights into the molecular mechanisms controlling the initiation of sperm motility we have used a phosphoproteomic approach, based on TiO2 enrichment of phosphopeptides from both immotile and motile spermatozoa collected from the cauda of rat epididymes. A quantitative proteomic analysis was used to evaluate the differential expression of phosphopeptides before and after the activation of motility. Intriguingly, two phosphorylation events occurred on Dynein Axonemal Intermediate chain 1 (Dnai1), including phosphorylation of ser124 and ser127. To understand what role these events play in sperm motility, we used the latest genetic technology, the CRISPR/Cas9 system, to generate a mutant mouse line where these potentially essential proteins could not undergo this phosphorylation event. Mice homozygous for a mutation in both ser124 and ser127 appear to show a slight reduction in their fertility when compared to heterozygous controls. Therefore we conclude that this phosphorylation event may be required for the acquisition of motility in spermatozoa.