Spiromesifen (SPM) is widely used for orchard mites and white fly control. The ecotoxicological data suggested that SPM is highly toxic to fish, but the information about its toxic effect on zebrafish is still obscure. In this study, adult zebrafish were exposed to SPM for 21 days. The plasma sex steroid hormone levels reflected the ratio of 17β-estradiol (E2) to testosterone (T) (E2/T) was significantly increased at 0.50 μg/L of SPM in male fish (2.4-fold, p = 0.049). Following 21 days' post exposure, distinct pathological changes were noted in gonad, males were more sensitive than female, which showed the interstitial connective tissue hyperplasia and widener in testis at 15 μg/L of SPM. In male fish, the relative percentage of spermatozoa was 13% decreased at 30 μg/L of SPM (p = 0.041). Which suggest SPM potential role in disrupting male gonad development. qRT-PCR results suggest that expression of follicle stimulating hormone receptor (fshr) was significantly down regulated in female zebrafish (0.29 fold of control, p = 0.010). Variable importance of projection (VIP) scores indicate the most important features separate in female and male. The different response of steroid level towards SPM between male and female zebrafish may due to the distinct regulation of key genes related in steroidogenesis and metabolism. This study for the first time connects the biochemical and histological to reveal the adverse effects of SPM on adult zebrafish in a sex dependent manner.