Background: To study the immunomodulatory and protective role of dietary administered purified β-glucan obtained from edible mushroom (Pleurotus florida) in commercial broiler chicken, experimentally challenged with virulent Newcastle disease virus (NDV) on 7th day post treatment. Mushroom glucan (MG) at 15 mg/kg feed (group A) and MG at 30 mg/kg feed (group B) was administered to broiler birds for 20 days keeping control birds (group C) with a normal diet throughout. After 7 days post treatment, three groups of birds (n = 4, in each case) were challenged with virulent NDV. The immunological parameters were assessed to observe the protective efficacy of MG. Results: When compared to the treatment regime, it was observed that in all the cases, group B birds showed higher immune-cellular and humoral responses in terms of enhanced immune-effector activities of blood leucocytes and intestinal intra-epithelial leucocytes and antibody production besides protection against NDV challenge than the others. After NDV challenge, 100% mortality was observed in control birds within 4 days, whereas in treated birds 50% and 75% protection of challenged birds was observed in group A and group B birds, respectively. The superoxide anion production by blood leucocytes of group A (0.641 ± 0.01) and group B (0.721 ± 0.01) birds were significantly higher than the control birds (0.283 ± 0.04) when assessed on 4th day post challenge. Group A (27.33 ± 1.20 μl and 25.33 ± 2.02 μl) and group B (33.66 ± 0.33 μl and 32.66 ± 0.33 μl) birds showed higher in vitro nitrite production by peripheral blood mononuclear cells (PBMC) and intestinal intra-epithelial leucocytes (iIEL), respectively, than the control (14.00 ± 0.57 μl and11 ± 0.57 μl) after challenge with virulent ND virus. In vitro lymphoproliferation (expressed as stimulation index) was significantly high in PBMC and iIEL of group A (0.371 ± 0.02 and 0.295 ± 0.02) and group B (0.428 ± 0.01 and 0.314 ± 0.01), respectively, than control (0.203 ± 0.01 and 0.135 ± 0.01) on 4th day of NDV challenge.