The objective of this study was to evaluate effects of sodium-butyrate supplementation on gastrointestinal function and the inflammatory response to ruminal acidosis (RA) challenge in cows. Four nonlactating cows with a rumen cannula were assigned to two treatments in a crossover design. Treatments were ruminal administration of sodium-butyrate (BUT) or control (CON). Sodium-butyrate was provided as Gustor BP70 and administered at a butyrate dose of 0.04% per kg body weight. The CON premix was made by replacing sodium-butyrate with wheat bran. Experimental periods were 28 days long with 21-day washout period separating the treatments.On Day 25 of each period, corn starch was ruminally administered at 0.7% per kg body weight as RA challenge. After RA challenge, ruminal pH was lower, and endotoxin concentration was higher for cows provided with BUT than those with CON, but the increase in fecal starch and the decrease in fecal pH were attenuated by BUT. The effect of butyrate supplementation on serum lipopolysaccharide-binding protein after RA challenge was not found. From these findings, butyrate supplementation mitigated rectal acidosis by reducing the flux of fermentable carbohydrate into the large intestine. An anti-inflammatory effect of butyrate was not observed, possibly due to lower pH and higher endotoxin concentration in the rumen.