The cholesterol-lowering effect of Lactobacillus plantarum (L. plantarum) NCU116 on lipid metabolism of rats fed on a high fat diet was investigated. Sprague-Dawley rats were randomly divided into normal diet (ND) group, a high fat diet (HFD) group, HFD plus L. plantarum NCU116 groups with two different doses (NCU116-L, 108 colony forming units (CFU)/mL; NCU116-H, 109 CFU/mL). After treatment for 5 weeks, L. plantarum NCU116 had the potential ability to regulate lipid metabolism levels, morphology of liver and adipose tissues. In addition, the bacterium significantly improved gene expression of low-density lipoprotein (LDL) receptor and cholesterol 7α-hydroxylase (CYP7A1). These results suggest that L. plantarum NCU116 was able to alter lipid metabolism and reduce the cholesterol level, in particular, in the rats on a high fat diet through regulating gene expression of key factors relating to LDL receptor and CYP7A1.