Cdk5 and p35 are integral components of the sperm tail outer dense fibers (ODFs), which contribute to the distinct morphology and function of the sperm tail. In this study, we sought to characterize and investigate the significance of Cdk5/p35 association with ODFs. We show that ODF2 interacts with Cdk5 and p35 but not with the Cdk5/p35 heterodimer. By using deletion mutants, the ODF2 binding region in p35 was mapped to residues 122 to 198. This overlaps the Cdk5 binding region in p35, explaining the inability of ODF2 to bind to the Cdk5/p35 complex. In vitro phosphorylation assay showed that although Cdk5/p35 does not phosphorylate ODF2, it phosphorylates ODF1. Mass spectrometry revealed that Cdk5/p35 specifically phosphorylates Ser193 in the ODF1 C-terminal region containing the Cys-X-Pro motif, the interaction site for the novel RING finger protein, ODF1 interacting protein (OIP1), a candidate E3 ubiquitin ligase, that also localizes in the sperm tail. Cdk5 phosphorylation of ODF1 Ser193 results in enhanced ODF1-OIP1 interaction. These findings suggest that Cdk5 may be important in promoting ODF1 degradation, and potentially, the detachment and fragmentation of the sperm tail following fertilization.