Back ground: Optimal IVF and embryo culture conditions depend on the composition of culture media being the most important determinants of successful in vitro interaction of the gametes and subsequent embryo development. ATP is fundamental factor to maintain the life, by providing energy, and controlling the cell function and metabolism. Objective: To investigate the effect ATP supplied to culture medium on fertilization rate and in mice as model for human being. Materials and Methods: The female mice (90) were superovulated using superovulation program (SOP) to produce large number of oocyte were divided into three groups, G1 inseminated with vassal sperm in vitro alone (control) , G2 inseminated with vassal sperm in vitro using SMART enriched with ATP(1.25mM). While, G3 inseminated with vassal sperm in vitro using SMART medium enriched with ATP (2.5mM). Mouse spermatozoa were collected from both vas deferens. Then sperm parameters were assessed after 30 min. IVF technique was performed for 3 groups. Results: The results of the present study appear, significantly increment (P<0.05) in the IVF (%) was observed when using 2.5 mM ATP as compared to 1.25 mM ATP and control group. Conclusions: The result showed that addition of high ATP concentration (2.5 mM) enriched to the culture medium improved percentage of in vitro fertilization.