The objective was to evaluate whether long-term addition of 3-nitrooxypropanol (NOP) to a beef cattle diet results in a sustained reduction in enteric CH4 emissions in beef cattle. Eight ruminally cannulated heifers (637 ± 16.2 kg BW) were used in a completely randomized design with 2 treatments: Control (0 g/d of NOP) and NOP (2 g/d of NOP). Treatments were mixed by hand into the total mixed ration (60% forage, DM basis) at feeding time. Feed offered was restricted to 65% of ad libitum DMI (slightly over maintenance energy intake) and provided once per day. The duration of the experiment was 146 d, including an initial 18-d covariate period without NOP use; a 112-d treatment period with NOP addition to the diet, divided into four 28-d time intervals (d 1 to 28, 29 to 56, 57 to 84, and 85 to 112); and a final 16-d recovery period without NOP use. During the covariate period and at the end of each interval and the end of the recovery period, CH4 was measured for 3 d using whole animal metabolic chambers. The concentration of VFA was measured in rumen fluid samples collected 0, 3, and 6 h after feeding, and the microbial population was evaluated using rumen samples collected 3 h after feeding on d 12 of the covariate period, d 22 of each interval within the treatment period, and d 8 of the recovery period. Average DMI for the experiment was 7.04 ± 0.27 kg. Methane emissions were reduced by 59.2% when NOP was used (9.16 vs. 22.46 g/kg DMI; P < 0.01). Total VFA concentrations were not affected (P = 0.12); however, molar proportion of acetate was reduced and that for propionate increased when NOP was added (P < 0.01), which reduced the acetate to propionate ratio (3.0 vs. 4.0; P < 0.01). The total copy number of the 16S rRNA gene of total bacteria was not affected (P = 0.50) by NOP, but the copy number of the 16S rRNA gene of methanogens was reduced (P < 0.01) and the copy number of the 18S rRNA gene of protozoa was increased (P = 0.03). The residual effect of NOP for most of the variables studied was not observed or was minimal during the recovery period. These results demonstrated that the addition of NOP to a diet for beef cattle caused a sustained decrease of methanogenesis, with no sign of adaptation, and that these effects were reversed once NOP addition was discontinued