Elevated levels of atmospheric CO2 (eCO2) promote rice growth and increase methane (CH4) emissions from rice paddies, because increased input of plant photosynthate to soil stimulates methanogenic archae. However, temporal trends in the effects of eCO2 on rice growth and CH4 emissions are still unclear. To investigate changes in the effects of eCO2 over time, we conducted a two-season pot experiment in a walk-in growth chamber. Positive effects of eCO2 on rice leaf photosynthetic rate, biomass, and grain yield were similar between growing seasons. However, the effects of eCO2 on CH4 emissions decreased over time. Elevated CO2 increased CH4 emissions by 48%–101% in the first growing season, but only by 28%–30% in the second growing season. We also identified the microbial process underlying the acclimation of CH4 emissions to atmospheric CO2 enrichment: eCO2 stimulated the abundance of methanotrophs more strongly in soils that had been previously exposed to eCO2 than in soils that had not been. These results emphasize the need for long-term eCO2 experiments for accurate predictions of terrestrial feedbacks.