Abstract: To explore the effects of different pre-transport acclimation methods on water quality, biochemical parameters and tissue structure of juvenile Ictalurus punctatus during transportation, and to provide references for transportation of juvenile I. punctatus, we conducted pre-transport intensive acclimation and temperature acclimation. The pre-transport intensive acclimation involved fish-to-water ratios of 1∶2, 1∶3 and 1∶4, and the temperature acclimation was at 14, 19 and 24 ℃. The simulation transport lasted for 18 h, and we investigated the changes in water quality (Ammonia nitrogen, pH), biochemical parameters Glucose (Glu)、cortisol (Cor), lactate dehydrogenase (LDH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) as well as organizational structure (Skin and intestine) at different time (0^th, 2^nd, 6^th, 18^th hour) and 24-hour recovery after transport. The results show that pre-transport intensive acclimation with a fish-to-water ratio of 1∶4 and a temperature of 19 ℃ had lower ammonia nitrogen levels than the other treatments (P<0.05). Glu, Cor, LDH, CAT, SOD and MDA levels were higher than the other groups, and there was minimal damage to the skin and intestinal structures. In conclusion, pre-transport intensive acclimation with a fish-to-water ratio of 1∶4 and a temperature of 19 ℃ can improve anti-stress capacity effectively during transportation of juvenile I. punctatus.