Background: Dietary protein requirements are dependent on a variety of factors and water temperature is one of the most important abiotic factors affecting protein requirement of fish. This study was, therefore, conducted to investigate effects of water temperature on dietary protein requirement of fry Heteropneustes fossilis which has high demand in most of the Asian markets. Methods: Quadruplicate groups of 30 fish per treatment (2.97 ± 0.65 cm; 5.11 ± 0.34 g) were fed seven isoenergetic diets (17.9 kJ g −1 gross energy; 14.99 kJ g −1 digestible energy) containing dietary protein levels ranging from 28 to 52% at two water temperatures (18 and 26°C). Experimental diets were fed to apparent satiation as semi-moist cakes thrice daily at 17:00, 12:00, and 17:30 h for 12 weeks. For precise information, various growth parameters, protein deposition, hematological parameters, metabolic enzymes, and stress response were analyzed, and effects of water temperature on dietary protein requirement was recommended on the basis of response from above parameters. Results: Groups held at 26°C attained best growth, feed conversion, and protein deposition at 44% dietary protein indicating that temperature affected dietary protein requirement for optimum growth of H. fossilis fry and protein requirement seems to be satisfied with 44% dietary protein. Interestingly, interactive effects of both dietary protein levels and temperature were not found (P > 0.05). Fish reared at 18°C had comparatively higher values for aspartate and alanine transferases than those reared at 26°C water temperature which exhibited normal physiological value for these enzymes indicating that body metabolism was normal at this temperature. Hematological parameters also followed same pattern. Furthermore, fish reared at 26°C water temperature exhibited more resistant to thermal stress (P < 0.05). The 95% maximum plateau of protein deposition data using second-degree polynomial regression analyses exhibited dietary protein requirement of fry H. fossilis between 40.8 and 41.8% of diet at 26°C water temperature. The recommended range of dietary protein level and protein/digestible energy ratio for fry H. fossilis is 40.8-41.8% and 27.21-27.88 mg protein kJ −1 digestible energy, respectively. Conclusions: Information developed is of high significance for optimizing growth potential by making better utilization of nutrient at 26°C and, to develop effective management strategies for mass culture of this highly preferred fish species.