Maintaining genome integrity in germ cells is crucial for fertility and species survival. However, the DNA repair mechanisms that sustain genome integrity in primordial germ cells (PGCs), which cope with high levels of replication stress, remain largely unknown. While the TatD family of proteins, evolutionarily conserved nucleases, has been found to play roles in various DNA-related processes, their in vivo functions in vertebrates have yet to be fully elucidated. TATDN2 has recently been implicated in resolving R-loops and participating in the replication stress response in BRCA1-deficient cancer cells. Here we found that tatdn2 exhibits conserved expression in mitotic and early meiotic germ cells across teleosts and mammals. Using medaka fish as a model, we then showed that loss of tatdn2 leads to all-phenotypically male adults and infertility due to PGC depletion during mitotic proliferation. We further demonstrated that knockout of tatdn2 increases R-loop accumulation and DNA damage, subsequently triggering apoptosis in PGCs. These findings indicate that tatdn2 plays a critical role in DNA damage repair associated with R-loop resolution in mitotic PGCs. Our study provides novel insights into the physiological function of TATDN2 and the mechanisms of genome maintenance in PGCs.