In karst desertification (KD) regions, surface water (SW) easily enters underground through pore fissures and sinkholes despite the presence of abundant precipitation. Such regions have a typical distribution of “soil above and water below”, and, thus, the unique “karst drought” occurs. Hence, an urgent and primary problem in combating KD is to reach highly efficient utilization of water resources in these regions. We selected three karst research areas with different levels of karst desertification and different geomorphic types. By monitoring the storage and transformation of five types of water in the agroforestry system—precipitation, SW, groundwater (GW), soil water (SoW), plant water (PW), the following results were obtained: (1) In KD regions, a positive correlation was found among available precipitation, rainfall, and land evapotranspiration (LE), and LE was approximately equivalent to soil evaporation. (2) To varying degrees, agroforestry brings ecological benefits, including reducing surface runoff, increasing soil infiltration, lowering the transpiration rate, and reducing soil evaporation, thus achieving efficient use of water resources. (3) From 100 % rainfall, the transformation rates of SW, GW, PW, and SoW reached 0.14–12.71 %, 9.43–30.20 %, 9.79–49.97 %, and 40.72–82.58 %, respectively, and SoW showed a larger reserve than the other three types. (4) Drought stress contributes to the improvement of water use efficiency (WUE). Affected by drought stress, WUE was found to be the highest in a medium-intensity karst desertification environment. The transformation mechanisms of the five types of water observed in the agroforestry system provide a reference for efficient utilization of water resources in KD regions as well as theoretical support for addressing karst drought. They are also essential in helping to advance the ecological derivative industry, boosting the economy in karst mountainous areas, and controlling karst desertification.