The aim of this study was to evaluate the effects of diets with different starch concentrations and fish oil (FO) supplementation on lactation performance, in vivo total-tract nutrient digestibility, N balance, and methane (CH4) emissions in lactating dairy cows. The experiment was conducted as a 4 × 4 Latin square design with a 2 × 2 factorial arrangement: 2 concentrations of dietary starch [low vs. high: 23.7 and 27.7% on a dry matter (DM) basis; neutral detergent fiber/starch ratios: 1.47 and 1.12], the presence or absence of FO supplement (0.80% on a DM basis), and their interaction were evaluated. Four Italian Friesian cows were fed 1 of the following 4 diets in 4 consecutive 26-d periods: (1) low starch (LS), (2) low starch plus FO (LSO), (3) high starch (HS), and (4) high starch plus FO (HSO). The diets contained the same amount of forages (corn silage, alfalfa and meadow hays)