Listeria monocytogenes is the causative agent of human listeriosis, a potentially fatal foodborne infection. Clinical manifestations range from febrile gastroenteritis to more severe invasive forms including meningitis, encephalitis, abortions, and perinatal infections. This Gram-positive facultative intracellular pathogen has evolved multiple strategies to face extracellular innate defense mechanisms of the host and to invade and multiply intracellularly within macrophages and nonphagocytic cells. This chapter provides an updated panorama of recent advances in the characterization of L. monocytogenes virulence determinants in the postgenomic era.