The main objective of this study was to evaluate correlative relationships between rumen lipopolysaccharide (LPS) and mediators of acute phase response with milk fat yield and efficiency in dairy cows challenged with graded amounts of barley grain in the diet. An additional aim of the study was to quantify the intercow variation in relation to milk fat production and acute phase response in cows fed graded amounts of grain. Eight primiparous, lactating Holstein cows (60 d in milk) were assigned to 1 of the 4 total mixed rations containing barley grain at 0, 15, 30, and 45% (dry matter basis) in a replicated 4 x 4 Latin square design. Free rumen LPS, plasma acute phase proteins, and milk fat content were quantified in multiple samples collected on d 5 and 7 of the measurement periods shortly before the morning feeding. Results showed markedly greater concentrations of rumen LPS with increasing dietary grain level. The correlative analysis revealed strong negative relationships between rumen LPS and milk fat content and yield. The predictor variable of rumen LPS explained 69% of the variation during the milk fat reduction of the cows. The stronger depression in milk fat percentage was obtained when rumen LPS exceeded a threshold of 5,564 ng/mL, corresponding to a milk fat content of 3.39%. The increase in concentration of rumen LPS was also associated with declines in milk fat yield and 3.5% fat-corrected milk (R(2) = 0.50), as well as milk energy efficiency (R(2) = 0.43). The correlative analysis also indicated that the increase of plasma C-reactive protein (CRP) in response to higher grain feeding was associated with a linear decrease of milk fat content and yield (R(2) = 0.28 to 0.46). Furthermore, the statistical analysis revealed high percentages of intercow variation related to milk fat variables, as well as the responses of rumen LPS and plasma CRP. Taken together, the current results implicate rumen LPS and the host CRP response in the lowering of milk fat content and milk energy efficiency in dairy cows fed high-grain diets. Further research is warranted to understand the mechanism(s) by which rumen LPS and inflammatory responses to LPS lower milk fat synthesis and milk energy efficiency and to develop novel strategies for their prevention.