Aquaporins are water selective channels which play important roles in cell volume regulation during the transmission of spermatozoa to female tract. This study investigated the expression of aquaporin3 and determined the role of aquaporins in human sperm motility and mitochondrial membrane potential (MMP). RT-PCR and flow cytometry analysis were done to investigate aquaporin3 expression levels, and immunolocalisation of aquaporin3 in the spermatozoa was detected using immunocytochemical analysis. The sperm suspension was divided into four groups of spermatozoa: (a) Spermatozoa at 0 hr, (b) spermatozoa in control group, (c) spermatozoa treated with HgCl2 (as an aquaporin inhibitor) and (d) spermatozoa treated with HgCl2+ and 2-mercaptoethanol. The sperm samples were examined in terms of sperm motility and mitochondrial membrane potential. Results confirmed aquaporin3 expression in human spermatozoa and immunocytochemistry results showed an intense immunoreactivity in whole sperm tail. After 60 min, HgCl2 showed a significant decrease in motility and MMP compared to the control group. At this time point, 2-mercaptoethanol in the HgCl2+ 2-mercaptoethanol group reversed the effects of HgCl2 as compared to the HgCl2 group. Present study showed the expression and immunolocalisation of AQP3 in hu