The ubiquitous presence of fluoxetine (FLX) in aquatic environments poses great threat to fish species. However, little is known about its deleterious impacts on fish olfaction. In this study, the olfactory toxicity of FLX at environmentally realistic levels was assessed by monitoring the behavioral and electroolfactogram (EOG) responses to olfactory stimuli with goldfish (Carassius auratus), and the toxification mechanisms underlying the observed olfaction dysfunction were also investigated. Our results showed that the behavioral and EOG responses of goldfish to olfactory stimuli were significantly weakened by FLX, indicating an evident toxicity of FLX to olfaction. Moreover, FLX exposure led to significant alterations in olfactory initiation-related genes, suppression of ion pumps (Ca 2+ -ATPase and Na + /K + -ATPase), tissue lesions, and fewer olfactory sensory neurons in olfactory epithelium. In addition to altering the expression of olfactory transmission-related genes, comparative metabolomic analysis found that olfaction-related neurotransmitters (i.e., L-glutamate and acetylcholine) and the olfactory transduction pathway were significantly affected by FLX. Furthermore, evident tissue lesions, aggravated lipid peroxidation and apoptosis, and less neuropeptide Y were observed in the olfactory bulbs of FLX-exposed goldfish. Our findings indicate that FLX may hamper goldfish olfaction by interfering with the initiation, transmission, and processing of olfactory signals.