Vitamin B₆ or pyridoxine in the form of pyridoxal phosphate participates as a prosthetic group of enzymes in a large number of metabolic reactions, particularly those associated with the metabolism of proteins and amino acids. With the success of artificial propagation and larval production, the culture of cobia becomes widely distributed in southern coastal provinces of China(especially in Guangdong and Hainan Provinces) as well as Southeast Asia. However, currently the growth of cobia heavily depends on trash fish and the development of formulated feeds for cobia is still in its infancy. The objective of this study is to detect the optimal requirement of pyridoxine in diet of juvenile cobia Rachycentron canadum.Basal diet was formulated using vitaminfree casein, gelatin and fish protein concentrate as the protein source. The graded levels of PN(0,2,4,8,16 and 32 mg/kg diet) were added to the basal diets to formulate six experimental diets containing 0.22,1.89,3.87,7.54,14.75 and 29.88 mg PN/kg diet, analyzed by HPLC, respectively. Each diet was fed to three replicate groups of cobia in 300 L tanks for 9 weeks, and each tank was stocked with 25 fish ［initial weight （3.23±0.06） g］. The water salinity was from 30 to 34, temperature fluctuated from 28 to 32 ℃ and dissolved oxygen was above 7 mg/L. The results showed that specific growth rate(SGR) have an increasing trend with the increase of dietary pyridoxine(from 0.22 to 3.87 mg/kg), but no significant differences were observed among diets containing 3.87 mg/kg PN or above. The contents of PN and AST in liver of fish fed diets with 3.87 mg/kg were significantly higher than the treatments with the contents of PN lower than 3.87 mg/kg, however, there were no significant differences among diets containing 3.87-29.88 mg/kg PN. The contents of PLP and the activities of ALT of the diet containing 7.54 mg/kg PN were significantly higher than the diets containing PN<7.54 mg/kg, however, no significant differences were discovered among diets containing PN>7.54 mg/kg. The dietary pyridoxine requirement was estimated to be 3.09-3.26 mg/kg by the brokenline model based on the SGR, and the activities of ALT in fish liver.