Leptospira interrogans is responsible for the zoonotic disease leptospirosis. The pathogenic mechanisms of this spirochaete remain poorly understood; however, virulence has been correlated with increased phagocytic uptake and survival within macrophages. Leptospiral outer membrane proteins are thought to be responsible for persistence in vivo via interaction with specific host components. In this study, we analysed the transcriptional profile of a virulent strain and its culture-attenuated derivative strain to identify bacterial factors that may be involved in pathogenesis. Two outer membrane proteins, LMB216 and LigB (leptospiral immunoglobulin-like protein B) were downregulated more than 10-fold in the culture-attenuated strain. We show that both proteins play a role in leptospiral uptake by macrophages and that LMB216, as well as LigB, enhances the binding of leptospires to fibronectin. Taken together, our results indicate that LMB216 plays a role in pathogen interaction with host molecule/s, which may contribute to pathogenesis of leptospirosis.