Human sperm have been documented to display apoptosis-like features such as externalization of phosphatidylserine (EPS), disruption of the transmembrane mitochondrial potential (MMP) and activation of caspases. Our aim was to evaluate possible association between activation of the apoptosis cascade in human sperm and its oocyte penetration capacity using the zona free hamster oocyte penetration assay (SPA). Semen specimens from 76 unselected donors were subjected to double density gradient centrifugation followed by incubation under capacitating conditions for 3 h and SPA. Apoptosis signalling was monitored by assessment of EPS, disruption of MMP and activation of caspase-3 by flow cytometry. Semen samples with subnormal SPA values (<20% penetrated oocytes) contained significantly higher amounts of spermatozoa with EPS, disrupted MMP and activated caspase-3 compared with those samples with normal SPA values (>20% penetrated oocytes, p < 0.01). All three apoptosis markers showed a significantly negative correlation with the percentage of penetrated oocytes (p < 0.01). Apoptosis-related signalling appears to have a negative association with sperm–oocyte penetration. The exclusion of sperm presenting with those apoptosis-related features during assisted reproduction may improve success rates of procedures such as intrauterine insemination and in vitro fertilization.