As a typical triazole fungicide, difenoconazole is extensively used to control plant diseases; however, its residue in environmental waters poses a risk to aquatic organisms. In this study, we investigated the acute toxicity of different life stages and sub-lethal toxicity in embryonic yolk sac stage of difenoconazole to zebrafish, and the developmental toxicity in F1 generation of parents exposed to difenoconazole at different life stages of zebrafish. Furthermore, we used transcriptomics to explore the potential mechanisms of difenoconazole on the F1 larvae of parents exposed to the chemical at the embryonic stage. The results of this study showed that developmental defects were observed in the F1 embryo/larvae of parents exposed to 3, 30, and 300 μg/L of difenoconazole at different (embryo, larval, juvenile, and adult) life stages, and exposure to difenoconazole at the embryonic stage caused more severe developmental toxicity than those at other life stages. Developmental defects (malformation, inhibition of heartbeat and body length) were observed in the F1 embryos and larvae of parents exposed to difenoconazole at the embryonic stage. In addition, the total cholesterol and triglyceride contents were significantly reduced in the F1 larvae, and RNA-seq analysis revealed significant alterations in the expression of nine genes (msmo1, hsd17b7, sc5d, tm7sf2, ebp, cyp2r1, lss, cyp51, and cyp27b1) in the steroid synthesis pathway. This is suggested that F1 larvae of parents exposed to difenoconazole at the embryonic stage show abnormalities in the steroid biosynthetic pathway. These results reveal the differences in toxicity of difenoconazole to zebrafish at different life stages, improve studies on difenoconazole toxicity to zebrafish, and provide a new perspective for assessing the risk of contaminants to aquatic organisms.