Given the rising evidence that gut malfunction including changes in the gut microbiota composition, plays a major role in the development of obesity and associated metabolic diseases, the exploring of novel probiotic bacteria with potential health benefits has attracted great attention. Recently Lactobacillus spp., exert potent anti-obesity effects by regulating key transcriptional and translational factors in adipose tissues. However, the molecular mechanism behind the anti-obesity effect of probiotics is not yet fully understood. Therefore, we investigated the effect of Lactobacillus plantarum A29 on the expression of adipogenic and lipogenic genes in 3T3-L1 adipocytes and high-fat diet (HFD)-fed mice. We observed that the treatment of 3T3-L1 adipocytes with the cell-free metabolites of L plantarum inhibited their differentiation and fat depositions via downregulating the key adipogenic transcriptional factors (PPAR-γ, C/EBP-α, and C/EBP-β) and their downstream targets (FAS, aP2, ACC, and SREBP-1). Interestingly, supplementation with L plantarum reduced the fat mass and serum lipid profile concurrently with downregulation of lipogenic gene expression in the adipocytes, resulting in reductions in the bodyweight of HFD-fed obese mice. L plantarum treatment attenuated the development of obesity in HFD-fed mice via the activation of p38MAPK, p44/42, and AMPK-α by increasing their phosphorylation. Further analysis revealed that A29 modulated gut-associated microbiota composition. Thus, A 29 potential probiotic strain may alleviate the obesity development and its associated metabolic disorders via inhibiting PPARγ through activating the p38MAPK and p44/42 signaling pathways.