Almost nothing is known about the activities and diversities of microbial communities involved in As methylation in arsenic-rich shallow and deep sediments; the correlations between As biomethylation and environmental parameters also remain to be elucidated. To address these issues, we collected 9 arsenic-rich sediment samples from the depths of 1, 30, 65, 95, 114, 135, 175, 200 and 223 m in Jianghan Plain. Microcosm assay indicated that the microbial communities in all of the sediment samples significantly catalyzed arsenic methylation. We identified 90 unique arsM genes from the eight samples, all of which code for new or new-type ArsMs, suggesting that As-methylating microorganisms are widely distributed in the samples from shallow to deep sediments. To determine whether biomethylation of As occurs in the sediments under natural geochemical conditions, we conducted microcosm assays without exogenous As and carbons. After 80.0 days of incubation, approximately 4.5–15.5 μg L−1 DMAsV were detected in all of the microcosms with the exception of that from 30.0 m, and 2.0–9.0μg L−1 MMAsV were detected in the microcosms of 65, 135, 200, and 230m; moreover, approximately 36.4–97.2 μg L−1 soluble As(V) were detected from the nine sediment samples. This suggests that approximately 5.3%, 0%, 8.1%, 28.9%, 18.0%, 8.7%, 13.8%, 10.2% and 14.9% of total dissolved As were methylated by the microbial communities in the sediment samples from 1, 30, 65, 95, 114, 135, 175, 200 and 223m without exogenous As and carbons, respectively. The concentrations of biogenic DMAsV show significant positive correlations with the depths of sediments, and negative correlations with the environmental NH+ 4 and NaCl, but show no significant correlations with other environmental parameters, including NO 3, SO2+ 4, TOC, TON, Fe, Sb, Cu, K, Ca, Mg, Mn and Al. This work for the first time revealed the activities and diversities of As-methylating microbes in the arsenic-rich shallow and deep sediments, and helps to better understand the microorganisms-mediated biogeochemical cycles of arsenic in arsenic-rich shallow and deep sediments.