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Simple Summary: Mitigating methane emissions from ruminants requires strategies that are sustainable and acceptable to both consumers and producers. Direct-fed microbials could meet these requirements. Forty Holstein-Friesian cows were randomly allocated one of two treatments, a control or control plus a direct-fed microbial (MYLO®—a mixture of Lactobacillus species; Terragen Biotech Pty Ltd., Coolum Beach, Queensland, Australia). Adding the direct-fed microbial had no significant effect on feed intake, milk yield, feed conversion efficiency, or methane parameters. While these results are contrary to our expectations, all were numerically in a favorable direction. Given there are reports that diet and dose rate may impact the size of any effect, we recommend a dose–response study be undertaken using a basal diet that is commonly used in pasture-based dairy systems. Using direct-fed microbials to mitigate enteric methane emissions could be sustainable and acceptable to both consumers and producers. Forty lactating, multiparous, Holstein-Friesian cows were randomly allocated one of two treatments: (1) a base of ad libitum vetch (Vicia sativa) hay and 7.0 kg DM/d of a grain mix, or (2) the basal diet plus 10 mL of MYLO® (Terragen Biotech Pty Ltd., Coolum Beach, Queensland, Australia) delivering 4.17 × 108 cfu of Lactobacillus per mL. Neither feed intake (25.4 kg/d vs. 24.8 kg/d) nor milk yield (29.9 vs. 30.3 kg/d) were affected by treatment. Feed conversion efficiency was not affected by treatment when expressed on an energy-corrected milk basis (1.15 vs. 1.18 kg/kg DMI). Neither methane yield (31.6 vs. 31.1 g/kg DMI) nor methane intensity (27.1 vs. 25.2 g/kg energy corrected milk) were affected by treatments. While these results are contrary to our expectations and not significant, all were numerically in a favorable direction. Given there are reports that diet and dose rate may impact the size of any effect, we recommend a dose–response study be undertaken using a basal diet that is commonly used in pasture-based dairy systems. [ABSTRACT FROM AUTHOR]