This study investigated the effect of dietary methionine level on growth and lipid metabolism via the general control nonderepressible2 kinase (GCN2) pathway in cobia (Rachycentron canadum). Cobia were fed diets with six levels of methionine (0.62%, 0.84%, 1.02%, 1.15%, 1.25% and 1.42% of dry diet) with a constant cystine level (0.42% dry diet). The feeding experiment began in September 2013 and ended in December 2013; during the experiment, cobia were fed ad libitum twice daily (7:00 and 18:00h) for 10weeks. Cobia fed the diet with 1.02% methionine showed elevated weight gain (WG) and feed efficiency ratio (FER) compared with those fed the other diets (P<0.05). The content of liver lipid, total triglyceride, and total cholesterol were first enhanced significantly with increasing dietary methionine level from 0.62% to 1.02%, and then decreased markedly with higher levels of dietary methionine level (1.02% to 1.42%). Crude lipid was markedly elevated when the dietary methionine level was 1.02%, and then plateaued with higher dietary methionine level. The expression of genes associated with hepatic lipid synthesis (sterol regulatory element binding protein-1, peroxisome proliferator activated receptor γ, fatty acid synthetase, and stearoyl-CoA desaturase-1) were markedly up-regulated in fish fed the diet containing 1.02% methionine, whereas the transcriptional levels of lipolytic genes (peroxisome proliferator activated receptor α, carnitine acyl transferase-1, and lipase lipoprotein lipase) were elevated in fish fed the methionine-deficient diet (0.62%; P<0.05). The expression of insulin-like growth factor-I (IGF-I) was suppressed by the methionine-deficient diet, whereas the hepatic mRNA expression levels of genes related to amino acid responses (AAR), i.e., GCN2, activating transcription factor 4 (ATF4), CCAAT enhancer binding protein β (C/EBPβ), and asparagine synthetase (ASNS), were significantly up-regulated. In conclusion, the dietary methionine requirement of cobia was estimated to be 1.04% and 1.15% of dry matter (2.23% and 2.45% dietary protein) on the basis of WG and FER, respectively. Results of this study suggested that methionine deficiency could suppress growth, decrease lipid content, and inhibit expression of IGF-I and some genes related to lipid synthesis in cobia; these changes might be regulated by inducing the expression of genes related to the GCN2 pathway (GCN2, ATF4, C/EBPβ, and ASNS). Statement of relevanceThe present study was conducted to investigate the effect of dietary methionine on growth performance, plasma biochemical indexes, lipid content and gene expression involved in lipid metabolism and GCN2 pathway in cobia (Rachycentron canadum). Our findings have showed that methionine deficiency could suppress growth, decrease lipid content and inhibit expressions of IGF-I and some lipid synthesis related genes of cobia, which may be regulated by inducing the mRNA expressions of GCN2 pathway related genes (GCN2, ATF4, C/EBPβ and ASNS). The results are reliable and of both theoretical and practical importance.The work described has not been submitted elsewhere for publication, in whole or in part, and all the authors listed have approved the manuscript that is enclosed. I have read and have abided by the statement of ethical standards for manuscripts submitted to Aquaculture.