► Hepatocytes from Neotropical fish Prochilodus lineatus were isolated with dispase. ► Cyanotoxin cylindrospermopsin CYN reduced hepatocytes viability at low concentrations. ► Multixenobiotic resistance mechanism (MXR) was impaired by CYN. ► CYN increased ROS production and lipid peroxidation in hepatocytes. ► Protein and DNA damages as well as GSH/GSSG ratio were not involved in CYN toxicity. Cylindrospermopsin is a potent toxicant for eukaryotic cells produced by several cyanobacteria. Recently, primary hepatocyte cultures of Neotropical fish have been established, demonstrating to be a quite efficient in vitro model for cellular toxicology studies. In the current study, a protocol for culture of Prochilodus lineatus hepatocytes was established and utilized to investigate the cellular responses to purified cylindrospermopsin exposure. Hepatocytes were successfully dissociated with dispase, resulting in a cell yield of 6.36×107cellsg−1 of liver, viability of 97% and attachment on uncoated culture flasks. For investigation of cylindrospermopsin effects, hepatocytes were dissociated, cultured during 96h and exposed to three concentrations of the toxin (0.1, 1.0 or 10μgl−1) for 72h. Cylindrospermopsin exposure significantly decreased cell viability (0.1 and 1μgl−1) and multixenobiotic resistance mechanism, MXR (all exposed groups), but increased reactive oxygen/nitrogen species levels (all exposed groups) and lipid peroxidation (10μgl−1). On the other hand no significant alterations were observed for other biochemical biomarkers as 2GSH/GSSG ratio, protein carbonyl levels and DNA strand breaks or glutathione S-transferase and glucose 6-phosphate dehydrogenase activities. In conclusion, hepatocytes might be made sensitive to cylindrospermopsin, at least in part, due to reduction of xenobiotics and endobiotics efflux capacity by MXR. Additionally, the toxin exposure suggests important issues regarding hepatocytes survival at the lowest cylindrospermopsin concentrations.