Pomegranate seed oil (PSO) mainly consisted of punicic acid (PunA), has gained attention due to rich conjugated linolenic acids and potential application in the improvement of various diseases. The aim of this study was to explore the ability of PunA to modulate lipid metabolism associated with non-alcoholic fatty liver disease (NAFLD) and explore the emerging role of the gut microbiota in modulating hepatic lipid metabolism. In this regard, PunA’s capacity was evaluated through C57BL/6J fed with a choline and L-amino acid defined, high fat diet (CDAHFD). Mice were randomly divided into four groups: NC group, MC group, PSO group and Cur group. Collected results indicated that supplement of PunA improved hepatic lipid profile. PunA administration also ameliorated hepatic steatosis and lipid accumulation. mRNA analysis showed that PunA exerted ameliorative effect on hepatic lipid accumulation by downregulating the mRNA expression of Dgat1, Scd-1, Srebp-1c, but increasing expression level of Cpt-1α. Metagenomic analysis discovered that PunA increased species diversity and richness of intestinal microbiota, promoted proliferation of beneficial commensals and restraining the endotoxin-producing microorganisms in cecum of CDAHFD-administrated mice. Furthermore, PunA suppressed lipid accumulation and lipid droplet formation in a cell NAFLD model in vitro. Overall, PunA exhibited anti-lipogenic and lipid-lowering effects, improvement of intestinal flora. In summary, PunA could be a candidate for the exploitation of functional foods or nutraceuticals aiming NAFLD.