AbstractThe aim of this study was to provide a reference value for the safe regulation and control of ammonia nitrogen in the aquaculture of Sepia pharaonis. The effects of acute and chronic toxicity of ammonia on the cuttlefish, S. pharaonis, were tested experimentally using juvenile S. pharaonis. The results showed that the half‐lethal concentration (LC50 ) values of ammonia nitrogen in juvenile S. pharaonis with a body weight of 6.52 ± 0. 23 g at 24, 48, 72, and 96 h were 31.72, 25.77, 23.33, and 18.33 mg/L, respectively, and the corresponding un‐ionized ammonia nitrogen (UIA‐N) concentrations were 1.66, 1.35, 1.22, and 0.96 mg/L, respectively. Compared with the control, the survival rate, specific growth rate, and feed intake of juvenile S. pharaonis declined significantly, and the feed conversion ratio and hepatosomatic index increased significantly at 56 d after exposure to >1 mg/L ammonia nitrogen. Juvenile S. pharaonis should be maintained at a concentration of ammonia nitrogen of no more than 1 mg/L (UIA‐N is 0.056 mg/L) in culture, and removing harmful nitrogenous wastes from the seawater is critical in maintaining cuttlefish culture.