High grain feeding has been associated with ruminal pH depression and microbial dysbiosis in cattle. Yet, the impact of high grain feeding on the caprine rumen and hindgut microbial community and lipopolysaccharide (LPS) release is largely unknown. Therefore, the objective was to investigate the effect of increasing dietary levels of barley grain on the microbial composition and LPS concentrations in the rumen and colon of goats. Effects were compared with respect to the responses of ruminal and colonic pH and short-chain fatty acid (SCFA) generation. Growing goats (n = 5-6) were fed diets containing 0, 30, or 60% coarsely ground barley grain for 6 weeks. Ruminal ciliate protozoa were counted with Bürker counting chamber, and quantitative PCR was used to compare bacterial populations. Increasing dietary grain level linearly increased (P < 0.05) ruminal numbers of entodiniomorphids. With the 60% grain diet, there was a reduction in ruminal abundance of the genus Prevotella and Fibrobacter succinogenes, whereas the ruminal abundance of Lactobacillus spp. increased compared to the 0 and 30% grain diets (P < 0.05). In the colon, abundance of the genus Prevotella and F. succinogenes increased (P < 0.05) in goats fed the 60% grain diet compared to those fed the other diets. Colonic abundance of Clostridium cluster I was related to the presence of grain in the diet. Ruminal LPS concentration decreased (P < 0.05) in response to the 60% grain diet, whereas its colonic concentration increased in response to the same diet (P < 0.05). Present results provide first insight on the adaptive response of rumen protozoa and rumen and colonic bacterial populations to increasing dietary levels of grain in goats. Although luminal pH largely affects microbial populations, fermentable substrate flow to the caprine hindgut may have played a greater role for colonic bacterial populations in the present study.