Peroxisomal testis-specific 1 gene (Pxt1) is the only male germ cell-specific gene that encodes a peroxisomal protein known to date. To elucidate the role of Pxt1 in spermatogenesis, we generated transgenic mice expressing a c-MYC-PXT1 fusion protein under the control of the PGK2 promoter. Overexpression of Pxt1 resulted in induction of male germ cells' apoptosis mainly in primary spermatocytes, finally leading to male infertility. This prompted us to analyze the proapoptotic character of mouse PXT1, which harbors a BH3-like domain in the N-terminal part. In different cell lines, the overexpression of PXT1 also resulted in a dramatic increase of apoptosis, whereas the deletion of the BH3-like domain significantly reduced cell death events, thereby confirming that the domain is functional and essential for the proapoptotic activity of PXT1. Moreover, we demonstrated that PXT1 interacts with apoptosis regulator BAT3, which, if overexpressed, can protect cells from the PXT1-induced apoptosis. The PXT1-BAT3 association leads to PXT1 relocation from the cytoplasm to the nucleus. In summary, we demonstrated that PXT1 induces apoptosis via the BH3-like domain and that this process is inhibited by BAT3.