Our previous study showed that supplementation with a combination of Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 reduced the body weight, body fat percentage, body fat mass and L1 subcutaneous fat area in overweight subjects. We aimed to evaluate whether the changes in adiposity after supplementation with Lactobacillus strains were associated with metabolic intermediates. A randomized, double-blind, placebo-controlled study was conducted on 66 non-diabetic and overweight individuals. Over a 12-week period, the probiotic group consumed 2 g of probiotic powder, whereas the placebo group consumed the same product without the probiotics. To investigate metabolic alterations, we performed plasma metabolomics using ultra-performance liquid chromatography and mass spectrometry (UPLC-LTQ/Orbitrap MS). Probiotic supplementation significantly increased the levels of octenoylcarnitine (C8:1), tetradecenoylcarnitine (C14:1), decanoylcarnitine (C10) and dodecenoylcarnitine (C12:1) compared with the levels from placebo supplementation. In the probiotic group, the changes in the body weight, body fat percentage, body fat mass and L1 subcutaneous fat area were negatively associated with changes in the levels of C8:1, C14:1, C10 and C12:1 acylcarnitines. In overweight individuals, probiotic-induced weight loss and adiposity reduction from the probiotic supplementation were associated with an increase in medium-chain acylcarnitines.