The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin—a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%–26% and 33%–48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation.