Beta-defensin 20 (DEFB20) is widely expressed in the epididymis with gene features involved in epididymal sperm maturation. However, the action mechanism and function of DEFB20 in sperm maturation are still unclear. One of the important roles of beta-defensin is the ion channel activity. The cation channel sperm-associated protein (CatSper) alpha is an ion channel protein found on the sperm surface. This study aimed to investigate the interaction between DEFB20 and CatSper1-4 protein in relation to the sperm maturation process. Protein sequences were obtained from the National Center for Biotechnology Information (NCBI). Protein modeling and validation were carried out by using the Robetta modeling server and the Ramachandran plot method. Rosetta web server was used for the docking analysis. The results revealed a natural interaction between DEFB20 and CatSper1-4. The interaction occurred at the cation channel (close to the casein kinase II), ion transport protein, and kinase c phosphorylation of the CatSper1-4 active site. The DEFB20 region interacting with CatSper2-4 was the beta-defensin domain, while with CatSper1 was the non-beta-defensin domain. Based on the analysis, DEFB20 may interact with CatSper α subunits, particularly CatsSper1, to affect ion channel activity during sperm maturation.