The role of glutathione (GSH) and cysteine (Cys) conjugates in the detoxification of microcystin-LR (MC-LR) in bighead carp (Aristichthys nobilis) was examined under laboratory and field conditions. Wild individuals of bighead carp were collected from 5 eutrophic lakes along the Yangtze River, while in laboratory experiment, bighead carp were injected intraperitoneally with 500 μg purified MC-LR/kg body weight (bw). Contents of MC-LR and its glutathione (MC-LR-GSH) and cysteine conjugates (MC-LR-Cys) in the liver of bighead carp were determined by liquid chromatography electrospray ionization mass spectrum (LC-ESI-MS). In laboratory experiment, low concentrations of MC-LR-GSH (mean: 0.042 μg/g dry weight (DW)) were always detectable, and the mean ratio of MC-LR-Cys to MC-LR-GSH was 6.55. While, in field study, relatively high MC-LR-Cys concentration (mean: 0.22 μg/g DW) was detected, whereas MC-LR-GSH was occasionally detectable, and the average ratio of MC-LR-Cys to MC-LR-GSH was as high as 71.49. A positive correlation was found between MC-LR-Cys concentration in the liver of bighead carp and MC-LR content in seston from the five lakes (r = 0.85). These results suggest that MC-LR-Cys might be much more important than MC-LR-GSH in the detoxification of MC-LR in fish liver, and that cysteine conjugation of MC-LR might be a physiological mechanism for the phytoplanktivorous bighead carp to counteract toxic cyanobacteria.