Introduction: During spermiogendsis, sperm chromatin histones are replaced by protamine. After fertilization, the sprem nucleus is decondenced by glutathione (GSH). In this survey, we study the effect of various concentrations of GSH on human in vitro sperm decondensation. Material and Methods: After preparation and analysis of semen samples, sperm pellet was washed by Ham's F-10 medium twice and then the sperm solution was divided into Non - washed and washed groups. In each group 100 ml sperm solution mixed with 100 ml various concentrations of GSH (1, 5, 10, 15, 20, 40 and 80 mM) during 15, 30, 60, 90, and 120 minutes. In the control groups, Ham's F-10 added to sperm solution instead of GSH. Finally, nuclear chromatin decondensation (NCD) was assessed by using sodium dodecyl sulfate (SDS) method, which upon removal of spermolemma, allowes NCD to take place depending on chromatin status. Results: Non-washed group:: In this group 1 to 10 mM of GSH had low effects of sperm chromatin, which was not significant (P>0.05) and 15 and 20 mM of GSH had high significant effects on sperm which whereas 40 and 80 mM of GSH could induce NCD in high proportion of sperms of NCD. Washed group: In this group, 1 to 15 mM of GSH had low effects on sperm chromatin so that low proportion of sperms underwent NCD which was not significant (P>0.05) whereas the effect of 20 mM GSH of sperm chromatin was significant. In 40 and 80 mM of GSH high proportion of sperms were decondenced. Conclusion: GSH is able to induce NCD in vitro by crossing the spermolemma and it's effect is concentration dependent. Our result showed that 40 and 80 mM of GSH induced high proportion of sperms to NCD and if hyperstable sperms is affected by GSH, it might increase the fertilization rate.