Low-glycemic compared to high-glycemic diets have been shown to improve metabolic status and enhance fat oxidation. The randomized, double-blind, controlled intervention study aimed to evaluate the effects of an energy-reduced diet containing isomaltulose (ISO, Palatinose™) versus sucrose (SUC) on body weight loss. Sixty-four healthy overweight/obese adults were allocated to consume either 40 g/day ISO or SUC added to an energy-reduced diet for 12 weeks. Anthropometric measurements, body composition, and energy metabolism were assessed at baseline and after 4, 8, and 12 weeks. Fifty participants (age: 40.7 ± 11.7 y; BMI: 29.4 ± 2.7 kg/m²) completed the study. During the 12 weeks, both groups significantly lost weight (p < 0.001), which was more pronounced following ISO (−3.2 ± 2.9 vs. −2.1 ± 2.6 kg; p = 0.258). Moreover, for participants in the ISO group, this was accompanied by a significant reduction in fat mass (ISO: −1.9 ± 2.5, p = 0.005; SUC: −0.9 ± 2.6%, p = 0.224). The overall decrease in energy intake was significantly higher in the ISO compared to that in the SUC group (p = 0.022). In addition, breakfast containing ISO induced a significantly lower increase in postprandial respiratory quotient (RQ) (mean incremental area under the curve (iAUC)2h for ISO vs. SUC: 4.8 ± 4.1 vs. 6.9 ± 3.1, p = 0.047). The results suggest that ISO in exchange for SUC may help to facilitate body weight reduction, lower postprandial RQ associated with higher fat oxidation, and reduce energy intake.