We determined the impact of feed intake (DMI) and total-tract fiber digestibility (NDFd) on milk fatty acid (FA) profile and yield responses in dairy cows. Our analysis used individual observations (n = 808) from 169 Holstein cows from 11 studies. Diets (% DM) contained (mean ± SD) 30.1 ± 1.94 NDF, 26.9 ± 2.09 starch, and 4.12 ± 0.78 total FA. DMI averaged 27.2 kg/d (range 14 to 39 kg/d) and NDFd averaged 40.4% (range 28 to 58%). Milk FA were classified as: <16C (summation of FA lower than 16-carbon, de novo FA), 16C (summation of 16-carbon FA, mixed FA), >16C (summation of FA greater than 16-carbon, preformed FA), and are reported in g/100 g FA, g/100 g milk, and g/d. Mixed model regressions were developed taking into account experiment, period within experiment, and cow within experiment as random factors. Increasing DMI linearly increased <16C milk FA as g/100 g FA [17.6 ± 0.95 + 0.24 ± 0.02 × DMI, P < 0.001], g/100 g milk [0.25 ± 0.03 + 0.02 ± 0.002 × DMI, P < 0.001], and g/d [157 ± 5.89 + 7.57 ± 0.95 × DMI, P < 0.001]. Increasing DMI linearly decreased >16C milk FA as g/100 g FA [45.4 ± 1.43 – 0.21 ± 0.03 × DMI, P < 0.001] and g/100 g milk [31.3 ± 0.83 – 2.44 ± 0.37 × DMI, P < 0.001]; however, increasing DMI linearly increased >16C milk FA as g/d [451 ± 50.5 + 5.39 ± 1.20 × DMI, P < 0.001]. Increasing DMI was not associated with changes in 16C milk FA (P > 0.50). Increasing NDFd linearly increased <16C milk FA as g/100 g milk [0.70 ± 0.05 + 0.002 ± 0.0009 × NDFd, P = 0.05]; however, NDFd was not associated with <16C milk FA as g/100 g FA (P = 0.73) or g/d (P = 0.37). Increasing NDFd linearly increased 16C milk FA as g/100 g FA [32.5 ± 1.61 + 0.08 ± 0.02 × NDFd, P < 0.001], g/100 g milk [0.98 ± 0.06 + 0.005 ± 0.001 × NDFd, P < 0.001], and g/d [489 ± 39.0 + 1.39 ± 0.72 × NDFd, P = 0.05]. Increasing NDFd linearly decreased >16C milk FA as g/100 g FA [44.4 ± 1.73 – 0.09 ± 0.02 × NDFd, P < 0.001]; however, NDFd was not associated with > 16C as g/100 g milk (P = 0.97) or g/d (P = 0.41). Results demonstrate that DMI is important for predicting the concentration and yield of de novo and preformed milk FA, while NDFd effects on milk FA depend upon the unit that these FA groups are reported in. The interpretation of relationships between DMI and NDFd with milk FA must consider the unit that milk FA are reported in.