Aims: Probiotics and their metabolites (SCFA) can regulate energy homeostasis. The present study thus evaluates the synergistic effect of probiotic Enterococcus faecalis AG5 on HFD induced obesity and the role of propionic acid (PA) in apoptosis induction of 3T3-L1 pre-adipocyte. Methods: Male Wistar rats (n = 24) were used to develop an HFD induced obesity model for 24 weeks. The effect of the orally administered probiotic AG5 (18th–24th weeks, 1 × 109 CFU/ml) was evaluated using physiological, biochemical, anthropometry, histopathological and serological analyses. Apoptosis in 3T3-L1 pre-adipocyte was assessed using flow cytometry, protein expression of PPARγ, 5-LOX, NF-κB, p-AKT, caspase 10 and detection of caspase 3/7 by Immunofluorescence confirmed the apoptosis induced by PA. Key findings: Probiotic AG5 significantly reduced body weight, BMI, serum cholesterol, triglycerides (p < 0.05) and improved HDL, insulin and leptin but lowered LDL and VLDL (p > 0.05). An inflammatory response was reduced as evident by TNF-α IHC. AG5 reduced adipocyte hypertrophy and fatty acid accumulation. Flow cytometry confirmed late apoptosis in PA-AG5 and standard PA treated 3T3-L1 cells. 5-LOX inhibition is associated with apoptosis induction, and increased caspase 1p 10 is related to cell death initiation. The study initially showed a low PPARγ activity inhibiting 5-LOX which may relate to adipose apoptosis. Finally caspase 3/7 detection using immunofluorescence proved the role of PA in adipocyte apoptosis. Significance: The present study is a novel approach towards obesity mitigation involving adipocyte apoptosis. The role of SCFA in adipocyte apoptosis is very limited which can prove to be novel therapeutic approach in the future. © 2020 Elsevier Inc.