The intracellular bacterial pathogenCoxiella burnetiidirects biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supportsC. burnetiireplication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promoteC. burnetiiintracellular growth and PV expansion. We predict additionalC. burnetiieffectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predictedC. burnetiiT4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion byC. burnetiiduring infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termedCoxiellavacuolarprotein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins.C. burnetiiΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpEmutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpDand ΔcvpEmutants rescued intracellular growth and PV generation, whereas the growth ofC. burnetiiΔcvpBand ΔcvpCwas rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicateC. burnetiiencodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.