BACKGROUND: Silkworm pupa oil polyunsaturated fatty acid (SPO PUFA) has been confirmed to have a cholesterol-lowering function. METHODS AND RESULTS:The effect of SPO PUFA and its main component, -linolenic acid (ALA), on the metabolism of cholesterol and its regulation was investigated. The model of lipid denatured cells were constructed to carry out lipid accumulation, cholesterol metabolism and transformation. Real-time PCR and western blots were also used to analyze the expression levels of related genes and proteins to investigate the cholesterol efflux regulation mechanism. The data indicated that SPO PUFA and ALA dose-dependently decreased intracellular total cholesterol (TC) and enhanced total bile acid (TBA). They could also promote cholesterol removal by enhancing bile acid secretion and by upregulating genes LXR , PPAR , ABCA1, ABCG1, and CYP7A1, which were regulated by LXR /PPAR -ABCA1/ABCG1-CYP7A1 nuclear receptor signal pathways. CONCLUSIONS: This study is of great significance in maintaining the balance of cholesterol and lipid metabolism, and in reducing the risk of steatohepatitis.The effect of SPO PUFAs and ALA on related gene expression in L02 cells Total cellular RNA was lysed and harvested using the total RNA kit (Omega Bio Tek Inc., Norcross, GA, USA) according to the