Constructed treatment wetlands are efficient at retaining a range of pesticides, however the ultimate fate of many of these compound is not well understood. This study evaluated the effect of drain-fill cycling on the mineralization of chlorpyrifos, a commonly used organophosphate insecticide, in wetland sediment–water microcosms. Monitoring of the fate of 14C ring-labeled chlorpyrifos showed that drain-fill cycling resulted in significantly lower mineralization rates relative to permanently flooded conditions. The reduction in mineralization was linked to enhanced partitioning of the pesticide to the sediment phase, which could potentially inhibit chlorpyrifos hydrolysis and mineralization. Over the nearly two-month experiment, less than 2.5% of the added compound was mineralized. While rates of mineralization in this experiment were higher than those reported for other soils and sediments, their low magnitude underscores how persistent chlorpyrifos and its metabolites are in aquatic environments, and suggests that management strategies and ecological risk assessment should focus more on ultimate mineralization rather than the simple disappearance of the parent compound.