We and others have shown that the molecular signatures of diverse bacterial species accumulate in human atheromatous lesions. Here, we aimed to determine the effect of non-viable bacteria on foam cell formation in vitro. Using human monocyte-derived macrophages and the murine J774 macrophage cell line, we found that any of a diverse panel of heat-killed Gram-positive or Gram-negative bacteria shown previously to accumulate in human atherosclerotic lesions promoted marked induction of foam cell formation in macrophages, as assessed by light-microscopy of Oil-red-O stained cells and Nile-red-based flow cytometric quantification of cellular lipid accumulation. As Toll-like receptors (TLRs) have a central role in the induction of inflammatory signalling by bacteria, we next examined if specific TLR-ligands could also promote foam cell formation in the absence of intact bacteria. Remarkably, stimulation of macrophages with purified ligands specific for any of the TLRs (including lipopeptide, polyI:C, LPS, flagellin, ssRNA, loxoribine and CpG DNA) led to significant lipid accumulation. This process was not dependent on oxidation of low-density lipoprotein (LDL) as neither antioxidants nor the scavenger receptor blocker polyinosinic acid reduced foam cell formation. Moreover, the presence of LDL was not required for TLR-mediated foam cell formation. Specific inhibitors of TLR signalling prevented foam cell formation induced by TLR ligands or bacteria. We conclude that although the bacterial signatures present in human atheroma are likely to reflect non-viable, killed organisms, it remains possible that molecules derived from these organisms may promote the differentiation of macrophages to lipid-laden foam cells via mechanisms that are likely to include stimulation of TLRs.