The hypothesis was tested that butyrate presence in the digesta of distinct gastrointestinal tract (GIT) segments of broilers leads to differential effects on digesta retention time, gut morphology, and proteolytic enzymatic activities, ultimately resulting in differences in protein digestibility. A total of 320 male day-old Ross 308 broilers were randomly assigned to 5 dietary treatments: 1) control (no butyrate), 2) unprotected butyrate (main activity in the crop and gastric regions), 3) tributyrin (main activity in the small intestine), 4) fat-coated butyrate (activity in the whole GIT) and 5) unprotected butyrate combined with tributyrin, each replicated 8 times. Rapeseed meal was used in combination with a fine dietary particle size in order to challenge the digestive capacity of young broilers. Birds were dissected at 22, 23, and 24 d of age and samples of digesta at various GIT locations as well as tissues were collected. Butyrate concentration varied significantly across GIT segments depending on treatment, indicating that the dietary contrasts were successful. The apparent ileal digestibility of methionine tended to increase when butyrate and/or propionate was present in colonic and cecal contents, possibly due to modifications of GIT development and digesta transit time. Butyrate presence in the digesta of the crop, proventriculus and gizzard, on the contrary, decreased the apparent ileal digestibility of several amino acids (AA). In addition, butyrate presence beyond the gizzard elicited anorexic effect that might be attributable to changes in intestinal enteroendocrine L-cells secretory activities. The present study demonstrates that, in broilers, effects of butyrate on digestive processes are conditioned by the GIT segment wherein the molecule is present and indicates its influence on digestive function and bioavailability of AA.