The objective of this study was to assess the potential of 3-nitrooxypropanol, a novel methane inhibitor; and canola oil, a known methane mitigant; alone and in combination on methane emissions, rumen fermentation, and diet digestibility. Eight ruminally canulated beef heifers (Angus cross, 732 ± 43 kg) were used in a double 4 × 4 Latin square design with 4 28-d periods and assigned to one of 4 dietary treatments. The dietary treatments were: 1) control (CON) (no supplementation of 3-nitrooxypropanol or canola oil), 2) canola oil alone (OIL) (5% of diet DM), 3) 3-nitrooxypropanol alone (NOP; 200 mg/kg of diet DM; DSM Nutritional Products Ltd., Kaiseraugst, Switzerland), and 4) 3-nitrooxypropanol and canola oil combined (NOP+OIL). After a 14-d diet adaption, dry matter intake (DMI) was recorded daily. Rumen contents were collected on d 14 and 17 for volatile fatty acid (VFA) analysis and protozoal populations. Enteric methane emissions were measured on d 18 to 21 using open circuit chambers. Diet digestibility was measured on d 24 to 27. Methane production was reduced from 26.2 (CON) to 19.6, 17.9, and 12.7 g/kg of DMI, for OIL, NOP, and NOP+OIL, respectively (P < 0.01). Total VFA concentrations (mM) were greatest for CON (101.3), similar between OIL (94.8) and NOP (94.8), and lowest for NOP+OIL (88.3) (P < 0.01). A decrease in acetate (A) and increase in propionate (P) proportions, and therefore a decrease in the A:P ratio was also observed with the OIL, NOP, and NOP+OIL treatments compared with CON (P < 0.01). The OIL and NOP+OIL treatments had a reduction in protozoa counts and a reduction in DM, OM, NDF, and ADF digestiblities when compared with CON and NOP (4.43 × 10⁴ vs. 4.24 × 10⁵/ mL rumen fluid; 60.7 vs. 66.8%; 62.0 vs. 68.7%; 47.6 vs. 61.0%; and 46.5 vs. 60.0%, respectively) (P < 0.01). The data demonstrated that the addition of OIL and NOP are effective means of decreasing methane production, and the combination of both caused the greatest reduction of methane emissions in cattle.