Background: Irrational use and unsafe handling of pesticides in agriculture farming is a serious public health concern in developing countries including Nepal. This study was conducted to assess the practice of chemical pesticide use and associated factors along with the acute health symptoms experienced by farmers in Chitwan District, Nepal.Methods: This was a descriptive cross-sectional study using quantitative methods with 790 farmers from Chitwan district, Nepal selected through systematic random sampling. Scores for knowledge, attitude/perception and practice were calculated by aggregating variables where each variable was scored 1 or 0 based on a right or wrong answer. Further, the practice variable was segregated into three groups namely practice during purchasing, practice during mixing and spraying and practice during storage and disposal. Statistical analysis was performed with X2 test, T-test and Multiple Logistic Regression. Confounder controls were made for age, sex and education.Results: Out of the 790 farmers, 663 (84.0%) were found to use exclusively chemical pesticide. Among them, 62% had no idea about its label. Farmers with better knowledge on pesticide handling were 8.3 times more likely to practice safe purchasing (95% CI=5.0-13.8), four times more likely to practice safe mixing and spraying (95% CI=2.5-5.9) and two times more likely to safe storage and disposal (95% CI=1.6-3.6). Similarly, perception/attitude of farmers about chemical pesticide policy and market management was significantly associated with the practice of farmers during purchasing (95% CI=1.1-2.8), mixing and spraying (95% CI=2.0-4.5) and storage and disposal (95% CI=1.1-2.5). Farmers with unsafe practice of pesticide handling were two times more likely to suffer from acute poisoning (AOR = 2.1, 95% CI = 1.3-3.3).Conclusion: There was a high use of pesticide in agriculture farming in Chitwan District. Improving knowledge of farmers on safe handling of pesticides and developing strategies for proper market management would promote the safe handling practices among farmers and reduce the acute pesticide poisoning in Nepal.