Elevated blood cholesterol is an important risk factor associated with atherosclerosis and CHD. The search for mediators that fine tune cholesterol homeostasis has recognised probiotics as being potentially beneficial. Here, we present data describing bacterial regulation of Niemann-Pick C1-like 1 (NPC1L1), which, when weakly expressed, results in a marked reduction in intestinal absorption of cholesterol. The probiotic Lactobacillus acidophilus ATCC 4356 reduced NPC1L1 gene expression and inhibited the cellular uptake of micellar cholesterol in Caco-2 cells. Soluble effector molecules secreted by ATCC 4356 were shown to be responsible for the decrease in NPC1L1. Furthermore, ATCC 4356 mediated this effect partly through the liver X receptors (LXR). The role of NPC1L1 and the LXR in cholesterol metabolism underscores the basis for the use of probiotics, such as ATCC 4356, in managing hypercholesterolaemia.