The objective of the present study was to evaluate the effect of Lactobacillus acidophilus 4356 on cholesterol metabolism in vivo. Rats were fed a cholesterol-enriched experimental diet with or without L. acidophilus 4356 supplementation at a dose of 109 colony-forming units per d. L. acidophilus 4356 feeding significantly lowered total serum cholesterol, LDL-cholesterol and TAG concentrations, but there was no change in the serum HDL-cholesterol concentrations. In addition, total liver cholesterol and TAG were decreased in the L. acidophilus 4356-fed group. The expression of Niemann-Pick C1-Like 1 (NPC1L1) in the duodenum and jejunum was significantly decreased following L. acidophilus 4356 feeding. Lactobacillus acidophilus 4356 increased the population of lactobacilli and bifidobacteria in the small intestine and faeces compared with the control. These results indicate that the probiotic potential of the L. acidophilus 4356 strain in the control of cholesterol is at least partially mediated by the down-regulation of NPC1L1. Furthermore, these results also potentially suggest a new mechanism that is responsible for the cholesterol-reducing effects of probiotics.