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β-glucans are widely-known immunostimulants that are profusely used in aquaculture industry. The present study was conducted to evaluate the effect of different in-feed doses of β-1,3/1,6-glucans on the expression of antioxidant and stress-related genes (GST, HSP-70, Vtg), inflammation related genes (Il-8, TNFα, CXC-chemokine and CAS) and adaptive immune-related genes (MHC-IIβ, TLR-7, IgM-H, and Mx) of Oreochromis niloticus challenged and non-challenged with Streptococcus iniae. Six experimental groups were established: non-challenged control (non-supplemented diet), challenged control (non-supplemented diet), non-challenged supplemented with 0.1% β-glucan, challenged supplemented with 0.1% β-glucan, non-challenged supplemented with 0.2% β-glucan and challenged supplemented with 0.2% β-glucan. Fish were fed with β-glucan for 21 days prior challenge and then sampled after 1, 3 and 7 days post-challenge. In non-challenged group, variable effects of the two doses of β-Glucans on the expression of the studied genes were observed; 0.1% induced higher expression of HSP70, CXC chemokine, MHC-IIβ and MX genes. Meanwhile, 0.2% induced better effect on the expression of Vtg, TNF-α, CAS and IgM-H, and almost equal effects of both doses on GST and IL8. However, with the challenged group, 0.2% β-Glucans showed better effect than 0.1% at day one post challenge through significant up-regulation of GST, HSP, IL8, TNF-α, CXC, and MHC-IIβ, meanwhile, the effect of 0.1% was only on the expression of HSP70, MHC-IIβ, and TLR7 at day 3 post challenge. No stimulatory role for both doses of β-Glucans on the expression of almost all genes at day 7 post-challenge. We conclude that both doses of β-glucan can modulate the antioxidant, inflammation, stress and immune-related genes in Nile tilapia, moreover, 0.2% β-Glucans showed better protective effect with Streptococcus iniae challange.