Impaired maximal fat oxidation has been linked to obesity and weight regain after weight loss. The aim was to investigate the relationship between maximal fat oxidation (MFO) and long-term weight loss maintenance. Eighty subjects [means (SD): age, 36(13) yrs; BMI, 38(1) kg/m2] were recruited from a total of 2,420 former participants of an 11- to 12-wk lifestyle intervention. Three groups were established based on percent weight loss at follow-up [5.3(3.3) yr]: clinical weight loss maintenance (CWL), >10% weight loss; moderate weight loss (MWL), 1-10% weight loss; and weight regain (WR). Body composition (dual X-ray absorptiometry) and fat oxidation (indirect calorimetry) during incremental exercise were measured at follow-up. Blood and a muscle biopsy were sampled. At follow-up, a U-shaped parabolic relationship between MFO and percent weight loss was observed (r = 0.448; P < 0.001). Overall differences between CWL, MWL, and WR were observed in MFO (mean [95% confidence interval], in g/min, respectively: 0.46 [0.41-0.52]; 0.32 [0.27-0.38]; 0.45 [0.38-0.51]; P = 0.002), maximal oxygen uptake (V̇o2max, in ml·min-1·FFM-1, respectively; 49 [46-51]; 43 [40-47]; 41 [39-44]; P = 0.007), HAD-activity (in µmol·g-1·min-1, respectively: 123 [113-133]; 104 [91-118]; 97 [88-105]; P < 0.001), muscle protein content of CD36 (in AU, respectively: 1.1 [1.0-1.2]; 0.9 [0.8-1.0]; 0.9 [0.8-0.9]; P = 0.008) and FABPpm (in AU, respectively, 1.0 [0.8-1.2]; 0.7 [0.5-0.8]; 0.7 [0.5-0.9]; P = 0.008), body fat (in %, respectively: 33 [29-38]; 42 [38-46]; 52 [49-55]; P < 0.001), and plasma triglycerides (in mM, respectively: 0.8 [0.7-1.0]; 1.3 [0.9-1.7]; 1.6 [1.0-2.1]; P = 0.013). CWL and WR both had higher MFO compared with MWL, but based on different mechanisms. CWL displayed higher V̇o2max and intramuscular capacity for fat oxidation, whereas abundance of lipids at whole-body level and in plasma was higher in WR.NEW & NOTEWORTHY Impaired maximal fat oxidation has been linked to obesity and weight regain after weight loss. Noteworthy, maximal fat oxidation was equally high after clinical weight loss maintenance and weight regain compared with moderate weight loss. A high maximal fat oxidation after clinical weight loss maintenance was related to higher maximal oxygen updake, content of key proteins involved in transport of lipids across the plasma membrane and β-oxidation. In contrast, a high maximal fat oxidation after weight regain was related to higher availability of lipids, i.e., general adiposity and plasma concentration of triglycerides.