Abstract: This study was undertaken to monitor the effect of 0.1 mM Mn2+ on the glutathione s-transferase activity (GST) in nicotine treated and untreated spermatozoa samples and to calculate the Michalis-Menten kinetic parameter of the reaction. Semen samples were collected from healthy volunteer donors after semen analysis, sample was subjected for preparation of 10,000 g supernatant. The enzymatic activity has been performed using 1-chloro, 2-4 dinitrobenzene (CDNB) as a substrate. The 0.1 mM Mn2+ supplementation was found to decrease the GST activities significantly (p<0.001). CDNB-GSH conjugate formation were almost significantly (p<0.05) lowered down with the addition of 0.1 mM Mn2+ to nicotine treated samples. The substrate affinity constant (km) of the reaction was found to be 44.44 FM. It remained unchanged throughout the Vmax which was considerably lowered upon Mn2+ addition to the spermatozoal samples. The data was transformed to Lineweaver-Burk 1/v vs 1/[S] plot which indicate the behavior of this enzyme was non-competitive in nature.