This study investigated the effect of 3-nitrooxypropanol (3NOP), a methane (CH₄) inhibitor, on rumen fermentation, lactational performance, and reproductive function in Holstein dairy cows. The study was a 15-wk randomized complete block design with 56 multi- and primiparous cows (including 8 rumen-cannulated). Cows were blocked based on their previous lactation milk yield or predicted milk yield and within block were randomly assigned to one of 2 treatments: (1) control, no 3NOP, and (2) 3NOP applied at 60 mg/kg feed DM. Cows entered the study within 3 d after calving and remained on the study for 105 d. Enteric CH₄ emission was measured during experimental wks 2, 6, 9, and 15 using the GreenFeed system. Rumen samples for fermentation analyses were collected during wks 4, 8, and 12. Milk was sampled 3 d/wk from 2 to 8 wks after calving and assayed for progesterone concentration to determine onset of ovarian activity. Compared with the control, 3NOP decreased total VFA and acetate, increased butyrate molar proportion, ethanol, and formate (P ≤ 0.05), and tended (P = 0.14) to increase dissolved hydrogen concentrations in the rumen. CH₄ emission was decreased 25% by 3NOP (337 vs. 225 g/d for the control; P < 0.001). 3NOP also decreased (P < 0.001) CH₄ emission yield (14.0 vs. 11.2 g/kg DMI) and intensity (8.3 vs. 6.3 g/kg ECM). DMI was lower for 3NOP compared with the control (23.7 vs. 24.9 kg/d; P = 0.05), but DMI as % of BW was not different (4.0%; P = 0.94) between treatments. Treatment had no effect (P ≥ 0.63) on milk and ECM yields, averaging 44 kg and 41.7 kg/d, respectively. Feed and ECM feed efficiencies were increased (P ≤ 0.01) by 3NOP compared with the control. Milk composition and milk fat and protein yields were not affected (P ≥ 0.39) by treatment. There was no effect (P ≥ 0.14) of 3NOP on return to ovarian activity, days to first and second CL, and length of the first and second luteal phases. In this experiment, 3NOP decreased enteric CH₄ emission, yield, and intensity without affecting milk yield and composition or onset of ovarian activity in early lactation dairy cows.