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Aim: To study the effect of supplementation of different levels of selenium as nanoparticles/sodium selenite on blood biochemical profile and humoral immunity in male Wistar rats. Materials and Methods: The experimental research was conducted at Division of Animal Nutrition, Indian Veterinary Research Institute, Izatnagar. 63 male Wistar rats were divided into 9 equal groups on the basis of their mean body weight (BW) (124.3±3.1 g BW) following completely randomized design. Experimental feeding was similar in all the groups except for the source and level of selenium (Se) in the diet. While Group 1 (control) was fed a basal diet with no Se supplementation, in Groups 2 and 3, 150 ppb Se was supplemented either as sodium selenite or Se nanoparticles, respectively. In Groups 4, 5, 6 and 7, Se was supplemented as its nanoparticles at 50%, 25%, 12.5% and 6.25% levels respectively i.e. at 75 ppb, 37.5 ppb, 18.75 ppb and 9.375 ppb levels respectively. In Groups 8 and 9, 300 ppb Se was supplemented either as Se nanoparticles or sodium selenite, respectively. Experimental feeding was conducted for a period of 91 days. At the end of the experimental trial, blood samples were collected to analyze the blood serum biochemical profile (serum glucose, serum total protein (TP), serum albumin, serum globulin, serum albumin: globulin ratio [A:G ratio], serum total cholesterol) and humoral immunity. Results: The levels of serum glucose, serum TP and serum albumin were comparable (p>0.05) among the nine groups of male Wistar rats. The mean serum total cholesterol was significantly (p