Effective treatments of obesity focusing on energy expenditure (EE) are needed. To evaluate future EE-modulating drug candidates, appropriate animal models and methods to assess EE are needed. This study aimed to evaluate the stable isotope ¹³C-bicarbonate method (¹³C-BM) for estimating EE in Göttingen minipigs under basal and drug-treated conditions. Four experiments (Expt.1-4) were conducted to assess: 1) the ¹³C-BM reproducibility using breath sample collection (n = 8), on two consecutive days, 2) the effect of two dose levels (5 and 10 mg/kg body weight (BW)) of the mitochondrial uncoupler dinitrophenol (DNP) in a crossover design (n = 8), 3) sampling method agreement; blood vs. exhaled air (n = 6) and 4) ¹³C-BM using constant isotope infusion compared with indirect calorimetry (IC) (n = 3). Results correlated significantly (p < 0.001) between days (Expt.1), with an average coefficient of variance of 5.4 ± 2.3%. Administration of 10 mg DNP/kg BW increased (p < 0.01) EE by 33.2 ± 6.4% (Expt.2). Results based on different sampling methods correlated significantly (p < 0.001) and EE increased after 10 mg DNP/kg BW (p < 0.05) in Expt.3. However, results based on blood sampling were significantly higher (p < 0.01) than those of exhaled air. No effect of DNP and significantly different EE results (p < 0.05) was observed in a limited number of animals, when constant isotope infusion and blood sampling was compared with IC (Expt.4). In conclusion, the ¹³C-BM is useful for investigating treatment effects on EE in minipigs. However, further validation under standardized conditions is needed to provide accurate estimates of the ¹³C recovery factor and respiratory quotient, both of decisive importance when using the ¹³C-BM.