The objective of this study was to evaluate effects of butyrate supplementation on blood glucagon-like peptide-2 (GLP-2) concentration, nutrient digestibility and responses to SARA challenge of cows fed diets differing in starch content. Eight Holstein cows were blocked by parity and assigned to one of 4 × 4 Latin squares balanced for carryover effects with a 2 × 2 factorial arrangement of treatments. Treatments were dietary starch content [low starch = 20% (LS) vs. high starch = 29% (HS)] and butyrate supplementation (butyrate vs. control) with 21-d periods. Butyrate was provided as Gustor BP70 WS (NOREL, S.A., Madrid, Spain), containing 70% sodium butyrate and 30% fatty acid mixture, at 2% of dietary DM, and control premix contained 70% wheat bran and 30% fatty acid mixture. Feeds, orts, and fecal samples were collected from d 17 to 19 to determine apparent total-tract nutrient digestibility. Blood samples were collected on d 19. Cows were feed-restricted at 60% of daily intake on d 20, and SARA challenge was conducted by providing 0.6%BW of steam flake corn grain in addition to each treatment diet on d 21, and blood and ruminal fluid samples were collected. Data were analyzed using Fit model procedure of JMP. The model included fixed effects of dietary starch content, butyrate supplement, their interaction, period, and square, and random effects of cow nested in squares. Cows fed butyrate increased serum BHB concentration (P < 0.01), tended to increase plasma GLP-2 concentration (P = 0.06), and increased DM digestibility (P < 0.05) compared with control. During SARA challenge, rumen endotoxin concentration was higher for cows fed HS + butyrate compared with cows fed the other diets (P < 0.05), but cows fed butyrate tended to decrease plasma/rumen endotoxin ratio than control (P = 0.08). Serum haptoglobin concentration was not affected by treatment. These results indicate that butyrate supplementation may increase plasma GLP-2 concentration and total-tract DM digestibility, and keep plasma endotoxin concentration low relative to its ruminal concentration.