The aim of the study was to evaluate the effectiveness of soybean meal (SBM) substitution with isolated soya protein (ISP) in pre-starter diet on immune system and performance of broiler chickens. After sex identification, 200 male Ross 308 broiler chicks were randomly allotted to 4 treatment groups with 5 replicates, 10 birds in each, and fed for 35 days. Experimental treatments including 4 substitution levels of SBM with ISP (0, 10, 20 and 30%) were fed from 1 to 7 day of age. Then all birds were fed common grower (days 7–24) and finisher (days 24–35) commercial diets. Dietary ISP inclusion improved (P < 0.05) body weight gain and feed conversion ratio during days 1–7 and 1–24 of age. Dietary ISP supplementation increased (P < 0.05) European Production Efficiency Factor and protein efficiency ratio throughout the experiment. Dietary ISP inclusion did not have an effect on relative weight of immune organs at days 3 and 7 of age. Dietary ISP inclusion linearly increased total anti-sheep red blood cells (anti-SRBC) (P = 0.017), immunoglobulin (Ig) G (P = 0.021) and IgM (P = 0.003) at 35 day of age. So, the inclusion of ISP (20 or 30% substitution of the SBM) in the pre-starter diet may improve immune response and performance of broiler chickens.