Abstract Testicular injuries like torsion or cryptorchidism can cause massive germ cell death, which could have great impact on male reproductive health. In addition, it has been proposed that modern life style, in the form of underwear or sedentary work position, could increase the testicular temperature, induce germ cell apoptosis, reduce spermatozoa quality and promote male infertility. In this work we showed that a heat stress stimulus induced massive germ cells apoptosis, which was associated with p38 mitogen‐activated protein kinase (MAPK) phosphorylation along with an increase in the levels of mRNA encoding calpain 2. Synthetic calpain inhibitors prevented heat stress‐induced germ cell apoptosis through inhibition of p38 MAPK phosphorylation. Thus, our results indicate that the blockage of calpains suppresses p38 MAPK phosphorylation, and identifies calpain activation (most likely calpain 2) as an early event in heat stress‐induced male germ cell apoptosis. J. Cell. Physiol. 221: 296–305, 2009. © 2009 Wiley‐Liss, Inc.