Determining the optimal culture density is necessary to improve fish performance and profitability. A total of 1350 rainbow trout with an average initial weight of 30±1.5 g were obtained. Then, the fish were randomly distributed in 12 fiberglass tanks and reared at densities of 15, 30, 45 and 60 kg/m3 for 60 days. The final weight, weight gain, and protein efficiency ratio were significantly higher and the feed conversion ratio was lower in fish reared in densities of 30 and 45 kg compared to densities of 15 and 60 kg. The highest levels of serum cortisol and lactate were observed at the density of 60 kg, followed by the density of 15 kg, which indicates the presence of stress in high culture density and very low density. The activity of serum lysozyme in densities of 15 and 60 kg was significantly lower than the density of 30 kg. The lowest serum total antioxidant capacity value, osmolality, and complement activity (ACH50) and the highest serum ALT activity were observed in fish reared at the highest density. Serum globulin level was significantly higher and serum AST activity was lower at densities of 30 and 45 kg than the density of 60 kg. The lowest levels of serum cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), and triglycerides were also observed at the density of 60 kg. The results showed that very low culture density, like high culture density, can have negative effects on the performance of rainbow trout. Therefore, medium densities (30 and 45 kg/m3) are recommended for rearing of this species.