Gut bacteria may influence diet‐related obesity, partly via ANGPTL4, a circulating lipoprotein lipase (LPL) inhibitor that regulates fat‐storage in the adipose. Modulating gut flora for stimulatory activity towards ANGPTL4 may thus serve a protective function. We have previously shown that bioactives from Bifidobacterium sp. increased the levels of enterocytic ANGPTL4 in vitro. The goal of the present study was to investigate whether a synbiotic formulation containing probiotic Bifidobacterium sp. along with prebiotics inulin and galactooligosacharides can enhance ANGPTL4 in human subjects, by potentially enriching Bifidobacterium in their gut. 57 healthy overweight adults participated in this 12‐week parallel, double‐blind, placebo‐controlled pilot study, by adding the synbiotic or placebo to their habitual diet. Fecal samples, plasma samples, colonocytes and peripheral blood mononuclear cells (PBMCs) were collected from subjects at 0 and 12‐weeks for assessing changes in fecal microbiota, ANGPTL4 gene‐expression and plasma ANGPTL4. The synbiotic group demonstrated significant increases in Bifidobacterium numbers, ANGPTL4 gene‐expression in PBMCs, and plasma ANGPTL4 levels compared to placebo. Also, a significant positive correlation was seen between Bifidobacterium numbers and plasma ANGPTL4 levels in the synbiotic group. Bifidogenic diets may thus lower LPL‐mediated fat‐storage.