Heat stress results in apoptosis in testicular germ cells. A small heat shock protein (hsp), hsp32, is induced by heat stress in the testis, but little is known about its definitive function in physiological processes. To clarify the underlying role of hsp32, hsp32 expression and related signals in the heat shock pathway were analysed in mouse testes and Sertoli cells after heat stress in vivo and in vitro; meanwhile, expression of hsp32 was silenced only in the Sertoli cells using three different small interfering RNAs (siRNAs) to further verify the functional role of hsp32 in Sertoli cells, and hsp32-derived carbon monoxide (CO) contents in cultured media were analysed to clarify whether hsp32-derived CO involve in the apoptosis regulation mechanisms. The results from the in vivo experiment showed that the high expression levels of hsp32 (P < 0.05) were observed whether chronic, moderate or acute, transient heat exposure. The in vitro experiment showed that acute, transient heat exposure resulted in increases in Sertoli cells apoptosis (P < 0.01), the expression of hsp32 and caspase-3 activity; hsp32-siRNA knockdown of hsp32 expression resulted in upregulated apoptosis (P < 0.01) and caspase-3 activity (P < 0.01) in the Sertoli cells and hyperthermia increases CO (P < 0.01) release by Sertoli cells. The results suggested that upregulating hsp32 in Sertoli cells inhibits caspase-3 activity and alleviates heat-induced impairments in mouse testis; hsp32-derived CO may involve in the regulation mechanism.