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Objective: To determine in vitro spermatotoxic effect of diethanolamine on human spermatozoa.Methods:For this study, samples were collected from normal healthy donors. After liquefaction, samples were used for preparation of sperm suspension. Sperm suspension was incubated with different concentrations (100-500 μg/mL) of diethanolamine to evaluate sperm parameters such as sperm motility, sperm viability and sperm morphology. Statistical analysis was performed using analysis of variance (ANOVA) followed by Tukey’s test and the level of significance was accepted with P<0.05. Results: The results showed that diethanolamine caused significant decrease in sperm motility and sperm viability which was concentration and time-dependent. Microscopic analysis revealed concentration-dependent increase in various kinds of sperm morphological abnormalities. Conclusion: DEA is spermatotoxic which alters structure and function of human spermatozoa and may affect male reproductive health.