H2O2 induces apoptosis in variety of cells; however, the sensitivities of testicular germ cells to H2O2 are not known. In the present study, H2O2, at concentrations in the range 1–10 μm, was found to induce apoptosis in testicular germ cells in vitro. Following 1 h of treatment with 10 μm H2O2, a 10‐fold rise in the percentage of apoptotic cells was observed. Induction of germ cell apoptosis was directly associated with a significant (P < 0.01) increase in lipid peroxidation and a concomitant decrease in superoxide dismutase and catalase activity. Examination of apoptotic signalling pathways revealed an increased expression of extrinsic (Fas, FasL and caspase‐8) and intrinsic (Bid, Bak, Bad, Bax and caspase‐9) markers, as well as p53, along with a simultaneous decrease in the Bcl‐2 protein at the highest concentration of H2O2 exposure. Both, c‐jun N‐terminal kinase and p38 phosphorylated forms were found to be up‐regulated. Interestingly, up‐regulation of the nuclear transcription factor kappa B was also observed. The respective transcripts for many of the above proteins followed an identical trend. Caspase‐3 activity was also estimated to be 30‐fold higher. Taken together, the above data indicate that testicular germ cells are prone to apoptosis at very low concentrations of H2O2, the mechanism of which involves extrinsic and intrinsic as well other regulatory pathways.