β-galactomannans found in soy-based broiler feed are known to cause physiological effects that are hypothesized to be related to gut inflammation. Previous studies have shown that the incorporation of β-mannanase in the diet or as a supplement results in improvements to certain performance parameters related to gut health and feed conversion. Using kinome analysis, we characterized the mechanism of β-galactomannan activity and supplementation with β-mannanase on the gut of commercial broilers to understand the mode of action. Two doses of β-mannanase (200 and 400 g/ton of feed) with and without inclusion of additional β-galactomannan (3,000 ppm) were tested at 3 time points (d 14, d 28, and d 42 post hatch). Broilers were fed starter (d 0 to 14), grower (d 15 to 28), and finisher diets (d 29 to 42). Jejuna were collected from birds from each treatment condition and time point. Cluster analysis of the kinome data showed that birds clustered first by age, then predominantly by whether β-mannanase had been included in the diet. Biological pathway analysis showed that the inclusion of additional β-galactomannan into the diet resulted in increased signaling related to immune response, relative to our normal control diet (with reduced soybean meal). The addition of β-mannanase to the enhanced β-galactomannan diet eliminated the majority of this immune-related signaling, indicating that the feed-induced immune response within the jejuna had been eliminated by the addition of β-mannanase. We also saw changes in specific metabolic and gut function pathways in birds fed β-mannanase. These observed changes in β-mannanase-fed birds are likely the mechanism for the enhanced performance and feed conversion observed in birds given β-mannanase in their diets.