The aim of this experiment is to explore the effect of sodium sulfate (Na₂SO₄) on methane reduction in the rumen, and its impact on anaerobic methane-oxidizing archaea (ANME). Using mixed rumen fluid from four Angus cattle fistulas, this study conducted an in vitro fermentation. Adding Na₂SO₄ to the fermentation substrate resulted in sulfur concentrations in the substrate of 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, and 2.4%. The gas production rate and methane yield were measured using an in vitro gas production method. Subsequently, the fermentation fluid was collected to determine the fermentation parameters. The presence of ANME in the fermentation broth, as well as the relationship between the number of bacteria, archaea, sulfate reducing bacteria (SRB), ANME, and the amount of Na₂SO₄ added to the substrate, were measured using qPCR. The results showed that: (1) the addition of Na₂SO₄ could significantly reduce CH₄ production and was negatively correlated with CO₂ production; (2) ANME-1 and ANME-2c did exist in the fermentation broth; (3) the total number of archaea, SRB, ANME-1, and ANME-2c increased with the elevation of Na₂SO₄. The above results indicated that Na₂SO₄ could mitigate methane production via sulfate-dependent anaerobic methane oxidation (S-DAMO) in the rumen. In the future management of beef cattle, including sodium sulfate in their diet can stimulate S-DAMO activity, thereby promoting a reduction in methane emissions.