Heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) may be a key enzyme for the protection of cells against stress. Its anti-apoptotic effect has been attributed to its product, carbon monoxide (CO), in many types of cells. However, whether its anti-apoptotic mechanism plays a role in Sertoli cells (SCs) is not yet clear. We hypothesise that Hsp32/HO-1 and CO generated from it provide survival advantages in SCs by preventing apoptosis under heat exposure. After treatment of cultured SCs with hyperthermia and/or Hsp32/HO-1 activater hemin, apoptosis was measured valuated by annexin V-FITC and caspase-3 activation. We have also analysed the Hsp32/HO-1-derived CO content of cultured media and cyclic guanosine monophosphate (cGMP) production by enzyme-linked immunosorbent assay (ELISA). Hyperthermia induced SCs apoptosis, while preincubation with hemin suppressed SC hyperthermia-induced apoptosis. Hyperthermia and/or hemin increase Hsp32/HO-1 gene expression and the production of CO, which, in turn, stimulates the generation of cGMP. The results suggest that Hsp32/HO-1 is a protective factor in heat-stressed SCs, and that CO generated from Hsp32/HO-1 is involved in the anti-apoptotic pathway.