From early 21C, a carotenoid biosynthesis-inhibiting herbicide, picolinafen was utilized to suppress the growth of wheat or barley. Picolinafen is one of the worldwide-used herbicides because its residual effects prolonged only 9 days and the applicable range is not limited. However, some reports started to suggest that the picolinafen could be detected in the agricultural drainage and even the lakes or riverside. To verify the lethal effects and mechanism of picolinafen on aquatic ecosystem we used zebrafish embryos. The dose escalation treatment of picolinafen induced embryo death before hatching and malformation of survived embryos. Especially, the apoptotic cells were increased at anterior part including eyes. Also, picolinafen suppressed reactive oxygen species (ROS) generation for vasculogenesis in posterior part of survived embryos. The immature caudal vein plexus was detected in blood vessels of picolinafen treated embryos. In addition, angiogenetic regulatory factors were changed according to the treatment. Through this study, we discovered that carotenoid biosynthesis-inhibiting herbicide has toxic effects on the aquatic ecosystem.