Listeriolysin O (LLO) and ActA are essential virulence determinants for Listeria monocytogenes pathogenesis. Transcription of actA and hly, encoding LLO, is regulated by PrfA and increases dramatically during intracellular infection. The 5' untranslated regions (5' UTRs) of actA and prfA have been shown to upregulate expression of their respective gene products. Here, we demonstrate that the hly 5' UTR plays a critical role in regulating expression of LLO during intracellular infection. Deletion of the hly 5' UTR, while retaining the hly ribosome binding site, had a moderate effect on LLO production during growth in broth culture, yet resulted in a marked decrease in LLO levels during intracellular infection. The diminished level of LLO resulted in a significant defect in bacterial cell-to-cell spread during intracellular infection and a 10-fold reduction in virulence during in vivo infection of mice. Insertion of the hly 5' UTR sequence between a heterologous promoter and reporter gene sequences indicated that the hly 5' UTR functions independent of PrfA-mediated transcription and can enhance expression of cis-associated genes through a mechanism that appears to act at both a post-transcriptional and translational level. The ability of the hly 5' UTR to increase gene expression can be exploited to achieve PrfA-independent complementation of virulence genes and high-level expression of single copy heterologous genes in L. monocytogenes.