Rapid industrialization and urbanization release several chemicals such as phthalates into the environment and cause adverse effects on reproductive system, mainly endocrine disruption, testicular injury and decline in semen quality in humans. There are no reports in extrapolating of the epidemiological data with in vitro findings. Our study show the correlations between in vivo studies and in vitro data for the effect of phthalate esters. Healthy human males, in the age group 21 to 40 years, visiting Chhatrapati Sahuji Maharaj Medical University (CSMMU), Lucknow, as part of infertility investigation, were recruited as volunteers. Semen analysis was performed according to the WHO guidelines. Phthalate esters were analyzed by high-performance liquid chromatography (HPLC) and cell viability by MTT assay. In the in vitro studies, sperms were exposed to highest concentration in semen samples (5-10 times higher) for a period ranging between 30 min and 96 hours. An inverse relationship with sperm motility in epidemiological studies was concurrent by significant dose-and time-dependent decrease in the sperm motility under in vitro environment after 12-hour exposure. Cytotoxicity was observed only with the highest concentration after 96 hours of exposure. There are a significant correlation between phthalate ester diethylhexyl phthalate, di-n-butyl phthalate (DEHP and DBP) and sperm motility both in vitro and in vivo conditions. Additionally, in vitro experiments conducted not only adjunct to the existing in vivo data but also specify the effect of specific toxicants (DEHP and DBP) on sperm motility and viability. Results show the decrease in motility of sperms under in vitro conditions at the maximum range of in vivo measured levels and 5- or 10-folds higher to that found in human semen samples.