The aim of this experimental study was to evaluate the effects of nicotine on sperm motility and on nonconventional sperm parameters in vitro. Capacitated spermatozoa isolated from 10 normozoospermic, healthy, non-smoker men were evaluated. Spermatozoa were exposed to increasing concentrations of nicotine (0, 1, 10, and 100 nglml) for 3 and 24 hours. Progressive motility and the following nonconventional sperm parameters, evaluated by flow cytometry, were assessed: mitochondrial membrane potential, viability, phosphatidylserine externalization, late apoptosis, degree of chromatin compactness, and DNA fragmentation. Nicotine suppressed, in a concentration-dependent manner, sperm progressive motility starting from the lowest concentration used (1 ng/ml), Similarly, it reduced the percentage of viable spermatozoa and increased the number of spermatozoa in late apoptosis, with altered chromatin compactness, or DNA fragmentation already after 3 hours of incubation. These effects were observed at a concentration similar (100 nglml) to that found in the seminal plasma of smokers (70 ng/ml), with the exception of the effects on sperm DNA fragmentation whose significant effect was detected also at a lower concentration (10 ng/ml), Nicotine may be regarded as a noxious component of cigarette smoke on the male reproductive function.Cigarette smoking is a risk factor for the onset of male sexual and reproductive diseases even today despite this topic bring highly controversial. In particular, it seems to alter sperm function though the mechanism(s) are still not entirely clear. Indeed, the combustion of tobacco releases more than 4,000 chemicals; some of them in the gaseous phase (carbon monoxide, nitric oxide, ammonia, hydrocarbons, etc.), others, such as nicotine, in the particulate phase.Nicotine is an alkaloid, and in nature is found in a non-ionized form (lipophilic) in an alkaline environment and in an ionized form (hydrophilic) in an acid environment. Under physiological conditions (pH 7.3-7.5) because of its lipophilic form (31%), nicotine crosses the biological membranes and is absorbed by the mucous membranes and skin and more than 80% is metabolized in the liver, kidney, and lung. CYP2A6 is the main cytochrome involved in its metabolism and thus in its oxidation. Nicotine concentration in the saliva of a subject, who has just finished smoking a cigarette, is about 1.56 mg/ml, 100,000 times higher than concentration in the peripheral blood, while the concentration of nicotine absorbed is about 1 mg/cigarette. From the bloodstream, nicotine and its metabolites go into