Two closely related bacterial species, Segniliparus rotundus and Segniliparus rugosus, have emerged as important human pathogens, but little is known about the immune responses they elicit or their comparative pathophysiologies. To determine the virulence and immune responses of the two species, we compared their abilities to grow in phagocytic and non-phagocytic cells. Both species maintained non-replicating states within A549 epithelial cells. S. rugosus persisted longer and multiplied more rapidly inside murine bone marrow-derived macrophages (BMDMs), induced more pro-inflammatory cytokines, and induced higher levels of macrophage necrosis. Activation of BMDMs by both species was mediated by toll-like receptor 2 (TLR2), followed by mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) signaling pathways, indicating a critical role for TLR2 in Segniliparus-induced macrophage activation. S. rugosus triggered faster and stronger activation of MAPK signaling and IκB degradation, indicating that S. rugosus induces more pro-inflammatory cytokines than S. rotundus. Multifocal granulomatous inflammations in the liver and lung were observed in mice infected with S. rugosus, but S. rotundus was rapidly cleared from all organs tested within 15 days post-infection. Furthermore, S. rugosus induced faster infiltration of innate immune cells such as neutrophils and macrophages to the lung than S. rotundus. Our results suggest that S. rugosus is more virulent and induces a stronger immune response than S. rotundus.