The enantioselective bioactivity and acute toxicity to target and non-target model species, and environmental fate of isocarbophos (ICP) in rice cultivation were investigated systematically. Bioactivity and toxicity of S-(+)-ICP was significantly greater than R-(-)-ICP, and the difference was 2.9-101 times. Based on the toxic unit analysis, the toxic interaction of ICP enantiomers for target pests was synergistic effect, while for non-target fish was concentration addition or antagonistic effect. Rac-ICP displayed equivalent bioactivity to S-(+)-ICP under the equal dosage, but the toxicity of rac-ICP to the tested fishes reduced at least 2 times. Rac-ICP is more suitable than optically pure S-(+)-ICP for rice cultivation based on the toxicity and bioactivity results. In environmental behavior experiments, the main metabolite of ICP, isocarbophos oxon (ICPO) was detected in rice plants, water, rice and rice hull samples. S-( +)-ICP and S-(+ )-ICPO were more persistent than the R-form in these matrices. The comprehensive data of ICP enantiomers in rice cultivation will improve environmental and ecological risk assessment, and using racemate may be more safe and reasonable in rice cultivation system.