The Tonle Sap Lake in Cambodia is a dynamic flood-pulsed ecosystem that annually increases its surface area from roughly 2,500 km2 to over 12,500 km2 driven by seasonal flooding from the Mekong River. This flooding is thought to structure many of the critical ecological processes, including aquatic primary and secondary productivity. The lake also has a large fishery that supports the livelihoods of nearly 2 million people. We used a state-space oxygen mass balance model and continuous dissolved oxygen measurements from four locations to provide the first estimates of gross primary productivity (GPP) and ecosystem respiration (ER) for the Tonle Sap. GPP averaged 4.1±2.3 g O2 m−3 d−1 with minimal differences among sites. There was a negative correlation between monthly GPP and lake level (r = 0.45) and positive correlation with turbidity (r = 0.65). ER averaged 24.9±20.0 g O2 m−3 d−1 but had greater than six-fold variation among sites and minimal seasonal change. Repeated hypoxia was observed at most sampling sites along with persistent net heterotrophy (GPP<ER), indicating significant bacterial metabolism of organic matter that is likely incorporated into the larger food web. Using our measurements of GPP, we calibrated a hydrodynamic-productivity model and predicted aquatic net primary production (aNPP) of 2.0±0.2 g C m−2 d−1 (2.4±0.2 million tonnes C y−1). Considering a range of plausible values for the total fisheries catch, we estimate that fisheries harvest is an equivalent of 7–69% of total aNPP, which is substantially larger than global average for marine and freshwater systems. This is likely due to relatively efficient carbon transfer through the food web and support of fish production from terrestrial NPP. These analyses are an important first-step in quantifying the resource pathways that support this important ecosystem.