To investigate the effects of night temperature on sugar accumulation in watermelon fruit, fruits were treated with higher nighttime temperature under a greenhouse. The minimum nighttime ambient temperature of the heating box (18 °C) was ≈6 °C higher than that of the control. The heat-treated fruit weighed at the end of heating treatment, 16 days after anthesis (DAA), was greater than that of control, but fruit weight at harvesting, 42 DAA, was almost the same in both treatments. Cells of all portions of the heat-treated fruit at 16 DAA were much larger than those of the control, and cells in the outermost rectangular parallelepipeds (RPs; 15-mm long samples that were serially collected from a 10-mm thick disk along a 10-mm wide strip removed at the maximum transverse diameter of the fruit) of the heat-treated fruit were 80 μm or more larger than those of the control. At 16 DAA, the number of RPs with sucrose contents of 2 g·L−1 or more were six and nine in control and heat-treated fruit, respectively. At 42 DAA, content in the outer RPs of the heat-treated fruit was greater than that in the outer RPs of the control. The number of RPs with sucrose contents of 40 g·L−1 or more was five in the control and 11 in heat-treated fruit. Mean sucrose, glucose, and fructose in fruit at 16 DAA did not differ in the treated fruit from the control. However, the sucrose content of heat-treated fruit was 32% higher than that of the control at 42 DAA. Glucose and fructose content were lower in heat-treated fruit than in the control.