Feeding high-concentrate diets has the potential to cause milk fat depression, but several studies have suggested that dietary sugar can increase milk fat yield. Two experiments were conducted to evaluate the ability of dietary molasses to prevent milk fat depression in the presence of a 65% concentrate diet. In trial 1, molasses replaced corn grain at 0, 2.5, or 5% of diet dry matter in diets fed to 12 second-lactation Holstein cows (134±37 d in milk) in a 3×3 Latin square design. Trial 1 demonstrated that replacing up to 5% of dietary dry matter from corn with molasses had positive effects on de novo fatty acid synthesis, increasing the yield of short- and medium-chain fatty acids during diet-induced milk fat depression. Increasing inclusion rate of molasses increased milk fat concentration, but decreased milk yield and milk protein yield. Trial 2 used 7 ruminally cannulated, multiparous, late-lactation Holstein cows (220±18 d in milk) to evaluate effects of dietary molasses on ruminal parameters and milk composition, and also to assess whether increased metabolizable protein supply would alter these responses. Cows were randomly assigned to a dietary treatment sequence in a crossover split plot design with 0 and 5% molasses diets. Dietary treatments were fed for 28 d, with 16 d for diet adaptation, and the final 12 d for 2 abomasal infusion periods in a crossover arrangement. Abomasal infusions of water or AA (5 g of l-Met/d+15 g of l-Lys-HCl/d+5 g of l-His-HCl-H(2)O/d) were administered 3 times daily for 5 d, with 2 d between infusion periods. Administration of AA had no effect on concentration or yield of any milk components. Addition of molasses increased milk fat concentration (2.71 vs. 2.94±0.21%), but had no effect on yields of milk fat or protein. Dietary molasses decreased total volatile fatty acid concentration (141 vs. 133±4.6mM), decreased the molar proportion of propionate, and increased the molar proportion of butyrate in ruminal fluid. Molasses also increased ruminal pH (5.73 vs. 5.87±0.06), decreased the yield of trans-10 C181, and increased the yield of trans-11 C181 in milk fat. These data provide evidence that molasses may promote mammary de novo fatty acid synthesis in cows fed high-energy rations by moderating ruminal pH and altering ruminal fatty acid biohydrogenation pathways.