The carbon cycling of tropical ecosystems has received considerable attention over the last 1–2 decades; however, interactions between climate variation and tropical forest net ecosystem CO2 exchange (NEE) are still uncertain. To reduce this uncertainty, and assess the biophysical controls on NEE, we used the eddy covariance method over a 3 year period (2005–2008) to measure the CO2 flux and energy balance for a 25–28 m tall, mature tropical semideciduous forest located near Sinop Mato Grosso, Brazil. The study period encompassed warm‐dry, cool‐wet, and cool‐dry climate conditions, and based on previous research, we hypothesized that the net CO2 accumulation of the semideciduous forest would be lower during periods of drought. Using time series of the enhanced vegetation index (EVI), a NEE‐light‐use model, and path analysis, we found that the estimated quantum yield (a′, μmol CO2μmol photons−1) was directly affected by temporal variations in the EVI, precipitation, and photosynthetically active radiation (PAR), while the optimal rate of gross primary production (FGPP,opt, μmol m−2 s−1) was directly affected by the EVI and PAR. However, indirect effects of precipitation on the a′ and FGPP,opt were stronger than direct effects because variations in precipitation also lead to variations in the EVI and the atmospheric vapor pressure deficit (VPD). Daytime ecosystem respiration (FRE,day, μmol m−2 s−1) was directly affected by temporal variations in temperature and VPD and indirect effects of other variables were of lesser importance. Net ecosystem CO2 uptake was often higher in the dry season than the wet season, not because of a dry season “green‐up” but because rates of ecosystem respiration declined relatively more than rates of canopy photosynthesis. Over interannual timescales, average daily NEE increased over the 3 year study period and was highest in 2007–2008, which was also the driest year in terms of rainfall. However, 2007–2008 was also the coolest year during the 3 year study period, and the low temperature appeared to compensate for the low rainfall. Overall, our data suggest that the NEE of tropical semideciduous forests is sensitive to temporal variations in surface water availability but that indirect effects of other variables, such as temperature and VPD, are important in controlling CO2 gain and loss. Such interactions will be important for the future NEE under warmer and drier conditions that are anticipated with anthropogenic climate change.