To elaborate the seasonal variation of groundwater arsenic (As) and its underlying regulating biogeochemical processes, long term biogeochemical monitoring and sediment incubation experiments were established in the aquifers from the Jianghan Plain, central Yangtze River basin. The results indicated that the groundwater As concentration exhibited significant seasonal variations (exceeding 800 μg L−1) between the pre-monsoon (January, February and March) and monsoon (June, July and August) season, which was regulated by the redox status fluctuation in the groundwater. The correlation between the Fe2+ and As concentrations in the groundwater from different seasons suggested the As mobilization in the aquifers was controlled by the iron mineral transformation. Furthermore, the incubation experiments with the sediments collected from different depths in the same aquifers indicated that the variation of microbial community was correlated with the released As concentration (R = 0.7, P < 0.05) and the iron-reducing bacteria, including Pseudomonas, Clostridium and Geobacter, were the main drivers for the As mobilization from the sediments at 26 m and 36 m depth. In addition, the bacterial sulfate reducing process could affect the As temporal variation in groundwater through forming the Fe-sulfide minerals to scavenge the As in aqueous phase. Those results could provide new insights into the mechanism of As mobilization and seasonal As variation in groundwater systems.