Cigarette smoking by the male partner adversely affects assisted reproductive techniques, suggesting that it may damage sperm chromatin/DNA and consequently embryo development. The effects of graded concentrations of research cigarettes smoke extract (CSE) on motility, mitochondrial membrane potential (MMP), chromatin integrity and apoptosis were evaluated in spermatozoa obtained from 13 healthy, non-smoking men with normal sperm parameters, by flow cytometry. CSE suppressed sperm motility in a concentration- and time-dependent manner and increased the number of spermatozoa with low MMP, the main source of energy for sperm motility. In addition, CSE had a detrimental effect on sperm chromatin condensation and apoptosis. Indeed, it increased the number of spermatozoa with phosphatidylserine externalization, an early apoptotic sign, and fragmented DNA, a late apoptotic sign, in a concentration- and time-dependent manner. These effects of CSE were of similar or even greater magnitude to those obtained following incubation with tumour necrosis factor-alpha, a cytokine known for its negative impact on sperm function, used as positive control. Since transmission of smoking-induced sperm DNA alterations has been found in pre-implantation embryos, and this may predispose offspring to a greater risk of malformations, cancer and genetic diseases, men seeking to father a child are recommended to give up smoking.