The effect of water stress induced to enhance sugar accumulation in Satsuma mandarin (Citrus unshiu Marc.) fruit was investigated. Satsuma mandarin trees were subjected to water stress using mulch cultivation from late August to early December. In mulch treatment, soil was covered with double-layered plastic sheets that prevented rainfall from permeating the soil, but allowed water from soil to evaporate. The water status of soil, fine roots, pericarps, and juice vesicles was determined using the isopiestic psychrometer. As the severity of water stress increased, both water potential and osmotic potential of fine roots and pericarps significantly decreased in plants grown under mulch cultivation compared to well-watered trees. Although water potential and osmotic potential decreased, turgor of both roots and pericarps of the water stressed trees did not decrease under water stress conditions. Because turgor was maintained, osmoregulation occurred in Satsuma mandarin trees in response to water stress. The osmotic potential of juice vesicles in water-stressed fruit gradually decreased, and sugars accumulated in vesicle cells. Concentrations of sucrose, fructose, and glucose increased in fruit sap under water stress, and the acidity in the fruit juice increased. Furthermore, the total sugar content per fruit of water stressed trees was significantly higher than in fruit of well-watered trees. These results suggest that sugar accumulation in Satsuma mandarin fruit was not caused by dehydration under water stress but rather that sugars were accumulated by active osmoregulation in response to water stress. When sugar components in osmoregulated fruit were analyzed, it was found that monosaccharides, i.e., glucose and fructose, were largely responsible for active osmoregulation in fruit under water stress conditions.