Objectives: Chlorpyrifos (CPF) has been used heedlessly in India to protect crop plants from pest induced crop loss. The objective of the study was to examine the effect of CPF, a broad spectrum organophosphate pesticide, on the contraction of uterine smooth muscle in rat model. Methodology: Two graded doses of CPF were administered orally at a dose of 5.4 mg/kg.BW /d and 8.1mg/kg.BW /d to female Charles foster rats for 30 days. The record of the movement of the uterus was obtained with isotonic transducer coupled to RMS Polyrite D. The serum levels of estradiol and progesterone were measured as per standard protocols of kits of ERBA Diagnostic Gmbh Mannheim Germany. Results: We have observed a significant inhibition of the force and increase in frequency of the contraction of uterus in all phases of an estrous cycle dose dependently in CPF exposed groups of rats compared to control groups of rats. We have also observed significant decrease in the level of estradiol in a dose dependent manner in all test groups of rats. These results indicate that CPF impairs the contraction of the uterine smooth muscle probably by inhibiting the secretion of estradiol from ovarian follicles. Further, the oxytocin induced potentiation of the tonic tension of uterus was significantly counteracted by CPF. This result proves that CPF probably impairs the contraction of uterine smooth muscle by inhibiting the estradiol. Conclusion: In conclusion, CPF impairs the contraction of uterine smooth muscles by inhibiting the secretion of estradiol from ovary in rat.