The objective of this study was to evaluate effects of butyrate supplementation on DMI, milk production, and ruminal fermentation of lactating dairy cows fed diets differing in starch content. Eight Holstein cows in early lactation (58.6 ± 9.96 d in milk; mean ± SD), including 4 primiparous ruminally cannulated 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 (20 vs. 29%) and butyrate supplementation (butyrate vs. control) with 21-d periods. Experimental diets contained 36 and 30% corn silage, 18 and 15% grass silage, 46 and 55% concentrates, respectively for low starch (LS) and high starch (HS) diets, on DM basis. 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 (providing butyrate at 1.1% of dietary DM), and control premix contained 70% wheat bran and 30% fatty acid mixture. All data were analyzed using Fit model procedure of JMP with a model including fixed effects of dietary starch content, butyrate supplementation, their interaction, period, and square, and random effects of cow nested in squares. Interaction effects between dietary starch content and butyrate supplementation were not observed for primary response variables, and rumen pH and milk yield were not affected by treatment. However, cows fed butyrate increased milk fat content (4.58 vs. 4.37%; P = 0.04), milk fat yield (1.51 vs. 1.42 kg/d; P = 0.02), tended to increase 4% FCM yield (35.9 vs. 34.3 kg/d; P = 0.08) and feed efficiency (4% FCM/DMI; 1.56 vs. 1.50; P = 0.08), and decreased MUN concentration (10.8 vs. 11.7 mg/dL; P = 0.02) compared with control. Cows fed HS diets tended to increase DMI (23.3 vs. 22.5 kg/d; P = 0.06), increased yields of milk protein (1.13 vs. 1.05 kg/d; P < 0.01) and SNF (3.02 vs. 2.85 kg/d; P = 0.04), and decreased MUN concentration (10.3 vs. 12.2 mg/dL; P < 0.001). These results indicate that butyrate supplementation may increase milk fat production while high starch diets increase milk protein production.