Energy and water exchange between the surface and the atmosphere are important drivers to Earth’s climate from local to global scale. In this study, the energy dynamic and the biophysical mechanisms that control the energy partitioning over a natural grassland pasture over the Brazilian Pampa biome are investigated using two micrometeorological sites located 300 km apart, in Southern Brazil. The latent heat flux, LE, was the main component of the energy balance in both autumn-winter (AW) and spring-summer (SS) periods. Annually, approximately 60% of the available energy is used for evapotranspiration (ET). However, the Bowen ratio presents seasonal variability greater in AW than SS. Global radiation, Rg, is the atmospheric variable controlling LE and sensible heat flux, H. Hysteresis curves in the daily cycle were observed for ET and surface conductance, Cs, regarding the environmental variables, net radiation, vapor pressure deficit, and air temperature. Among the variables analyzed in the Pampa biome, surface conductance and evapotranspiration respond more strongly to the vapor pressure deficit. The hysteresis cycles formed by ET and conductance show a substantial biophysical control in the ET process. The results obtained here allowed a comprehension of the biophysical mechanisms involved in the energy partition process in natural grassland. Therefore, this study can be used as a base for research on land-use changes in this unique ecosystem of the Pampa biome.