Environmental factors influence the contents of taste components, such as capsaicinoid compounds, in the fruit of chili pepper (Capsicum annuum). The present research was conducted to evaluate the effect of water supply and harvesting date after flowering on sugar and capsaicinoid contents in fruit of the Japanese chili pepper cultivars ‘Botankosho’, ‘Fushimiamanaga’, ‘Manganji’, and ‘Sapporo Oonaga Nanban’. The experiment was conducted in a greenhouse from April to October in 2016 and 2017. Three water supply treatments were applied: 260 mL (excess), 130 mL (standard), and 50 mL (drought) per application. Fruit were harvested at 20, 30, 40, and 50 days after flowering (DAF). Glucose, glutamic acid, and total sugar were measured using a portable spectrophotometer, and capsaicinoid content was measured by HPLC. Total sugar content and Brix tended to increase with later harvesting, whereas glucose content did not change significantly by DAF. Sugars in the fruit were dominated by fructose, and the ratio of fructose content to total sugar content increased as the fruit matured. Glutamic acid content in the fruit increased up to 40 DAF, and thereafter remained unchanged or decreased. The capsaicinoid content of the fruit increased with fruit maturation. Elevation in water supply induced an increase in the fruit glucose content and decrease in total sugar and glutamic acid contents. Previous studies of tomato (Solanum lycopersicum L.) found that total sugar and glucose contents decrease in response to increase in water supply. Therefore, it is suggested that sugar metabolism and accumulation differ in the fruits of tomato and chili pepper. The highest capsaicinoid content in chili pepper fruit was observed in response to the drought treatment.