Present study was conducted to investigate the effects of heat-killed Tsukamurella inchonensis on growth performance, gastrointestinal structure, immune response, and biochemical parameters in rainbow trout. Fish (mean weight 25 g) were fed basal diet (control), diets containing 2.48 × 108 colony-forming units (low-dose group) or 1.24 × 109 colony-forming units (high-dose group) of heat-killed Tsukamurella inchonensis per 1 kg of feed for 90 days. Results showed that growth performance was significantly enhanced in both treatment groups compared to the control group. The intestinal villus length and pyloric cecal fold length were mainly enhanced in the high-dose group. On the other hand, higher goblet cell percentage was shown with administration of dead Tsukamurella inchonensis in both treatment groups. Immune parameters such as alternative complement activity, immunoglobulin level, and hemagglutination titer were significantly higher in treatment groups than in fish fed in the control diet. Meanwhile, feeding heat-killed Tsukamurella inchonensis especially at higher dose caused a decrease in the levels of total cholesterol, uric acid, and lipid peroxidation product whereas no significant changes were noted in serum-specific marker enzymes levels, namely alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) by feeding both treatment diets compared to the control group. This study suggests that heat-killed Tsukamurella inchonensis especially at 1.24 × 109 colony-forming units had more potential to enhance growth, immunological parameters, and intestinal structure in rainbow trout.