Organic matter (OM) plays an important role in the biogeochemical transformation of arsenic (As) in groundwater and sediments. High-As groundwater used for irrigation introduces dissolved As into the ground. Moreover, water table fluctuations and variations in redox conditions affect As mobilization in both the unsaturated and saturated zones. A field experiment was conducted at the DY site, Datong Basin, China to explore the effect of OM on As and iron (Fe) mobilization and transformation in the unsaturated and saturated zones during irrigation. Soil water/groundwater and sediments sampled at different depths during the experiment were analyzed to investigate the hydrochemical processes and correlations between As, Fe, and OM concentrations. Irrigation activities led to the accumulation of aqueous As in the saturated zone and increasing As content in the topsoil. Analyses of Fourier-transform infrared (FTIR) spectra suggest the existence of As-Fe-OM ternary complexation. Parallel factor analysis (PARAFAC) of the three-dimensional fluorescence excitation–emission matrix (EEM) reveals that water-extractable organic matter (WEOM) of sediments comprises three major components, two terrestrial humic-like components (C1 and C2) and one protein-like component (C3). And terrestrial humic-like components with high molecular weight promotes As-Fe-OM complexation reactions. The results of this study demonstrate As mobilization under irrigation conditions and contribute to a better understanding of the role of OM under high-As groundwater irrigation. This study has implications for controlling and managing OM or As-contaminated water for irrigation in high-As groundwater regions.