Herein, we examined emissions of CH4 and the community structures of methanogenic archaea and methanotrophic bacteria in paddy soils subjected to a novel irrigation system, namely continuous sub-irrigation with treated wastewater (TWW). This system has recently been developed by our group to effectively reuse TWW for the cultivation of protein-rich rice. The results showed that, despite not using mineral fertilisers, the wastewater reuse system produced a rice yield comparable to that of a conventional cultivation practice and reduced CH4 emissions from paddy fields by 80%. Continuous sub-irrigation with TWW significantly inhibited the growth of methanogens in the lower soil layer during the reproductive stage of rice plants, which was strongly consistent with the effective CH4 mitigation, resulting in a vast reduction in the abundance of methanotrophs in the upper soil layer. The compositions of the examined microbial communities were not particularly affected by the studied cultivation practices. Overall, this study demonstrated that continuous sub-irrigation with TWW was an effective method to produce high rice yield and simultaneously reduce CH4 emissions from paddy fields, and it also highlighted the potential underlying microbial mechanisms of the greenhouse gas mitigation.