The objective of this study was to investigate the effect of 3-nitrooxypropanol (3NOP), a methane inhibitor, on palatability of TMR for Holstein dairy cows. Following a 1-wk covariate period, 12 multiparous cows averaging (±SD) 74 ± 22 DIM, 53 ± 12 kg/d milk yield, and 630 ± 146 kg BW were fed a basal TMR containing increasing levels of 3NOP during 5, 6-d periods. The basal TMR (16.5% CP and 39.4% NDF) contained, on DM basis, 39% corn silage, 11%, alfalfa haylage, 4% grass hay, and 46% concentrate feeds and mineral-vitamin premix. Feed bins were split in half by a solid divider and cows simultaneously received the basal TMR supplemented with (1) a placebo (Period 1), or (2) 3NOP included in the TMR at 30, 60, 90, or 120 mg/kg DM (Periods 2–5). 3NOP was incorporated in the ration through a premix containing ground corn grain, soybean oil, and molasses; the inclusion rate of the premix was adjusted according to the targeted 3NOP concentration for each experimental period and DM of the TMR. Cows were fed twice daily: 60% of the daily feed DM allowance at 0800 h and 40% at 1800 h. Feed offered and refused was recorded at each feeding. During the morning feedings, each cow was offered either control or 3NOP-treated TMR at 150% of her normal intake. After collection of the evening refusals, cows received only the basal TMR without the premix until the next morning feeding. Location (left or right) of the control and 3NOP treated TMR within a feed bin was switched every day during each period to avoid feed location bias. Compared with the control (6.93 kg/10 h) DMI was increased (P < 0.001) by 3NOP inclusion rates of 30, 60, and 90 mg/kg DM (8.75, 8.78, and 9.35 kg/10 h, respectively). DMI was similar between control and 120 mg/kg 3NOP (7.23 kg/10 h). There was no effect of feed location (right vs left) within feed bin on DMI (P > 0.17). There was a treatment × day of TMR offering interaction (P < 0.001), which appeared to be due to a slight decrease over time in DMI with the 90 and 120 mg/kg 3NOP inclusion rates. In this short-term experiment, 3NOP did not affect DMI in lactating dairy cows.