AimsOverweight is a major global health problem. Various methodologies to get rid of the extra fat are available, but usually, those are associated with adverse side effects. Probiotics, on the contrary, seem to have the potential to help reduce fat accumulation without much apparent adversity. In this study, we have evaluated a pair of well-documented probiotics for their anti-obesogenic effects. Main methodsWe used strains of Lactobacillus acidophilus (LA) and a cocktail (LDB-ST) of Lactobacillus delbruckei sp. bulgaricus (LDB) and Streptococcus thermophilus (ST) in this study. The murine pre-adipocyte cell line 3T3-L1 was terminally differentiated to matured adipocytes to use as a model to evaluate the bacteria's anti-obesogenic effects. The optimal dose for treatment of both the probiotics was determined using a cell viability assay. We assessed the probiotic internalization potential of differentiated 3T3-L1 cells by flow cytometry, fluorescence microscopy, and cell lysis method. We determined the lipolytic and anti-adipogenic potential of probiotics by intracellular lipid staining, spectrophotometry, and gene expression analysis. Key findingsBoth probiotics were effective lipolytic agents as revealed by reducing cellular lipids and down-regulation of mammalian adipogenesis marker genes in terminally differentiated 3T3-L1 cells. SignificancePrevious studies from our group had proven the immune-modulatory properties of these probiotics on an immune-biased mouse model. The present study demonstrates LA and LDB-ST to be effective against adipogenesis. Further in vivo studies will be conducted to strengthen this claim.