Over 35 days feeding trial, the effects of lincomycin (LM) and butyrate glycerides (BG) supplementation on the growth performance, blood biochemical changes, immune response, and nutrient absorption related gene expression in broiler chickens were evaluated. A total of 480 one- day-old broiler chicks (Ross 308) were allotted into 6 groups (8 replicates/group with 10 birds/replicate). Experimental groups were arranged as follows; control group (A) fed the basal diet (BD); B, C, and D groups were fed the BD supplemented with 350, 500 and 1000 g BG/ton of feed respectively. While group E was fed the BD with 350 + 500 + 1000 g BG/ton of feed during starter, grower, and finisher periods, respectively, and finally group F which received lincomycin (LM) antibiotic (100 g/ton) in overall periods. Broilers that received BG in group E (birds supplemented with BG at 350 + 500 + 1000 g/ton feed) and LM (F) supplements both gained more body weight and weight growth (P<0.05). Lower feed intake and improved fcr were observed in all the experimental groups that received either BG or lm compared to control birds (P<0.05). Lower serum total cholesterol, triglycerides, and glutamic oxaloacetic transaminase (GOT) (P<0.05) were recorded in the lm or BG supplemented birds compared to the control. Higher immune response represented by enhanced antibody titer against H9N1, and newcastle disease (ND) and lower hepatic malondialdehyde (MDA) was obtained in groups D, E and F (P<0.05) compared to the other groups. Higher gene expression of GSH-Px, Glut 2, PepT1 and FAS was obtained in groups D, E and F (P<0.05) compared to the control. Supplemental BG or LM increased the unsaturated fatty acids (oleic, linoleic and linolenic acids) while lowered the palmitic acid contents in breast muscle (P<0.05) compared to control. In conclusion, supplementing broilers’ diets with BG or lincomycin improved their growth performance, immune response and muscle fatty acid profile as well as modulatory role in lipid metabolism. Supplementing starter, grower, and finisher broiler diets with BG at 350 + 500 + 1000 g/ton each may be a useful substitute for lincomycin.