Plant water use plays a crucial part in the soil-plant-atmosphere continuum. However, in karst regions, plants frequently suffer from water shortages due to low soil water storage capacity. Therefore, it is necessary to understand plant water consumption (as determined by sap flow) and seasonal variation of water sources to improve water management in karst catchments. In this study, thermal dissipation probes (TDP), calibrated using empirical equations, were used to measure the sap flow of three typical woody vegetations, including Coriaria nepalensis (sparse-shrub), Toona sinensis (secondary forest) and Populus adenopoda (shrub-grass). Oxygen and hydrogen stable isotopes were used to analyze seasonal variation of plant water sources. The results showed that: (1) T. Sinensis (3.89 ± 3.87 LÁday −1) had significantly higher daily sap flow than C. nepalensis (0.33 ± 0.37 LÁday −1) and P. adenopoda (0.09 ± 0.12 LÁday −1); (2) daily sap flow was closely correlated to photosynthetically active radiation (PAR) and vapour pressure deficit (VPD); (3) over the entire study period, plants mainly used water from the surface soil horizons; and (4) a greater proportion of epikarst water was used for C. nepalensis than by T. sinensis and P. adenopoda over the whole growth stage, and more epikarst water was used in early and mid-growth stages compared to the late stage for the three species. This study contributes to a deeper understanding of the plant water use strategies in karst regions, and is helpful for ecosystem management.