The objective of this study was to test the efficacy of Propionibacterium strains to mitigate enteric methane (CH4) emissions in beef heifers fed a mixed diet. An experiment was conducted with 16 ruminally cannulated beef heifers fed a basal diet consisting of 60 : 40 barley silage : barley grain (DM basis). Treatments included: (1) Control, (2) Propionibacterium freudenreichii T114, (3) P. thoenii T159, and (4) P. freudenreichii T54. Strains (1 × 1011 colony forming units) were administered daily directly into the rumen before feeding. No treatment effects were observed for DM intake (P = 0.90), mean ruminal pH (P = 0.50) and total volatile fatty acids (P = 0.44). However, compared with the Control, proportions of individual volatile fatty acids changed with acetate being less with Propionibacterium T159 (P = 0.02), whereas ruminal isobutyrate (P < 0.01) and acetate : propionate ratio (P = 0.04) were greater with Propionibacterium T114. Total daily enteric CH4 production averaged 188 g/day and was not affected by Propionbacterium strains (P = 0.51). Methane yield averaged 22 g/kg of DMI intake and tended to be greater with Propionibacterium strains (P = 0.08). The relative abundance of total Propionibacteria was greater with the inoculation of Propionibacterium T159 relative to the Control heifers (P = 0.04). In conclusion, inoculation of Propionibacterium T159 decreased ruminal acetate proportion and Propionibacterium T114 increased acetate : propionate ratio. However, inoculated strains failed to lower total CH4 emissions possibly due to the inability of Propionibacterium strains to elevate ruminal propionate concentrations.