Among all known mycotoxins, aflatoxin B1 (AFB1) has been one of the most studied for its hepatotoxic, carcinogenic, mutagenic, teratogenic and immunosuppressant effects. However, there have only been a few metabolic and toxicological studies on aflatoxins in farmed Sparus aurata and these have been restricted to in vivo trials. This study aimed to examine the effects of acute and chronic AFB1exposure on CYP1A and GST enzymes induced in vitro on S. aurata hepatocytes by immunoblot analysis, and to carry out apoptotic studies on the cytotoxic effects leading to cell death. Immunofluorescence analysis revealed that cell damage was not reversible but permanent, as the cellular repair systems were unable to recover from the induced toxic insult. We detected several CYP1A bands and showed an indirect correlation between induction of CYP1A with dose and time of exposure. The decrease in expression of CYP1A over prolonged exposure times, along with high toxic concentration, could be related to the lethal damage observed on hepatocytes under contrast phase and immunofluorescence analysis. A particular pattern of expression was found for GST isoforms upon AFB1 exposure, identifying two different kinds of toxic insult for each isoform profile. The 65 KDa and the 49 KDa bands were suggestive of markers of acute and chronic response, respectively. Interestingly, apoptosis induction, considered an early DNA lesion, was found to be associated with chronic damage along with low toxic concentration. The new cell model from S. aurata has been proven to be a useful and valid tool to be used in the further investigation of the modulated response of liver phase I and II enzymes to AFB1.