Coxiella burnetii is a Gram-negative pleomorphic bacterium and the causative agent of Q fever. During infection, the pathogen survives and replicates within a phagosome-like parasitophorous vacuole while influencing cellular functions throughout the host cell, indicating a capacity for effector protein secretion. Analysis of the C. burnetii (RSA 493 strain) genome sequence indicates that C. burnetii contains genes with homology to the Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS). T4BSSs have only been described in L. pneumophila and C. burnetii, marking it a unique virulence determinate. Characterization of bacterial virulence determinants ranging from autotransporter proteins to diverse secretion systems suggests that polar localization may be a virulence mechanism hallmark. To characterize T4BSS subcellular localization in C. burnetii, we analyzed C. burnetii-infected Vero cells by indirect immunofluorescent antibody (IFA) and immunoelectron microscopy (IEM). Using antibodies against the C. burnetii T4BSS homologs IcmT, IcmV, and DotH, IFA show that these proteins are localized to the poles of the bacterium. IEM supports this finding, showing that antibodies against C. burnetii IcmT and DotH preferentially localize to the bacterial cell pole(s). Together, these data demonstrate that the C. burnetii T4BSS localizes to the pole(s) of the bacterium during infection of host cells.