Inert gas is often used in the deoxygenation of microbial electrolysis cells (MECs) to maintain growth and viability of anaerobes. However, the effects of the gas atmosphere on hydrogen production and microbial community of MECs are often neglected. Here, the performances and biofilm microbiomes of MECs pre-sparged with different gases were compared. MECs pre-sparged with argon gas (Ar) yielded more hydrogen (3.73 ± 0.13 mol-H2/mol-acetate) and a higher hydrogen production rate (2.99 ± 0.17 L-H2/L-reactor-day) than MECs pre-sparged with N2 (3.41 ± 0.13 mol-H2/mol-acetate and 2.27 ± 0.28 L-H2/L-reactor-day, respectively). Microbiome analysis indicated that the relative abundance of Geobacter increased from 59.25% to 77.79% when the gas atmosphere in MECs shifted from N2 to Ar. Hydrogen production may have been catalyzed by nitrogenase from Geobacter and photosynthetic bacteria in MECs pre-sparged with Ar. These findings suggested that the gas atmosphere substantially influences the microbiome of anode biofilms and Ar sparging is most effective for enhancing hydrogen production in MECs.