This study was conducted to examine the effect of dietary natural compounds, such as β-glucan, carotenoids, oligosaccharides and anthocyanins, on immune response and tight junction proteins in broiler chickens. A total of 900 one-day-old chickens were allocated to five treatments in three floor pens (replicates) of 60 broilers per pen. Chickens were fed five diets: a control (basal) diet, a diet supplemented with β-glucan at 0.05%, or diets supplemented with carotenoids, oligosaccharides or anthocyanins at 0.5% of each compound. Male broilers were randomly selected for sample collections. On Day 25, plasma samples were collected from the brachial vein. On Day 26, six broilers were intraperitoneally injected with 2 mg of lipopolysaccharide per kg of body weight. Twelve hours later (Day 27), blood and ileum samples were collected to determine immune parameters and tight junction proteins using ELISA assays. The results showed that anthocyanin supplementation reduced the level of interleukin-1β compared to the lipopolysaccharide-injected control group (p = 0.047), which suggests that anthocyanin could partly alleviate the inflammation. Carotenoids reached a lower level of interleukin-6 compared to the β-glucan treatment (p = 0.0466). β-Glucan (p = 0.0382) and oligosaccharides (p = 0.0449) increased the level of plasma immunoglobulin G compared to the challenged control group, which may indicate an enhanced humoral immunity. Furthermore, β-glucan (except for occludin 2), carotenoids, oligosaccharides and anthocyanins increased (p <
0.05) the levels of ileal zonula occludens-1, occludin 1 and occludin 2 compared to the lipopolysaccharide-challenged control chickens. This may suggest that all the bioactive substances improved the gut barrier function. The plasma levels of tight junction proteins show higher concentrations in lipopolysaccharide-challenged groups compared to the non-challenged groups (p <
0.05). This may refer to the tight junction disruption and appearance in circulation as a reflection of lipopolysaccharide exposure.