Peroxisome proliferator-activated receptors (PPARs) are important targets for cannabidiol (CBD), which mediate many of its biological actions. The hypothesis of the present research assumed that PPARs affect the gut response to different challenge factors in chickens (C. perfringens vs. lipopolysaccharides (LPS) from E. coli), and that CBD can mediate the pathways of this response. The study proved that CBD and the challenge factors significantly affect the expression level of PPARα (p = 0.001) and selected genes determining gut barrier function. A positive correlation was demonstrated between PPARs and genes involved in the formation of tight junctions, immune, and oxidative stress responses in chickens. Dietary supplementation with CBD actively mediated the expression rate of PPARs, but the mechanism of interaction between CBD and PPARs was different depending on the stress factor used. The addition of CBD to the birds' diets did not contribute to reducing intestinal permeability under induced stress conditions nor cause stress, as indicated by the absence of elevated blood cortisol and endotoxin levels. CBD also supported the mechanisms of protecting intestinal cells from the cytotoxic effects in a C. perfringens challenge through the levels of genes involved in oxidative stress. This study indicates the importance of research toward understanding the mechanisms of PPARs as a target for enhancing intestinal barrier function, provides new results on the biological action of CBD in chickens, and shows a constant PPAR association with the jejunum mucosa of birds.