Leptospirosis, a bacterial zoonosis caused by pathogenic spp., is prevalent worldwide and has become a serious threat in recent years. Limited understanding of pathogenesis and host response has hampered the development of effective vaccine and diagnostics. Although is phagocytosed by innate immune cells, it resists its destruction, and the evading mechanism involved is unclear. In the present study, we used an integrative multi-omics approach to identify the critical molecular factors of involved in pathogenesis during interaction with human macrophages. Transcriptomic and proteomic analyses were performed at 24 h postinfection of human macrophages (phorbol-12-myristate-13-acetate differentiated THP-1 cells) with the pathogenic Leptospira interrogans serovar Icterohaemorrhagiae strain RGA (LEPIRGA). Our results identified a total of 1,528 transcripts and 871 proteins that were significantly expressed with an adjusted value of <0.05. The correlations between the transcriptomic and proteomic data were above average ( = 0.844), suggesting the role of the posttranscriptional processes during host interaction. The conjoint analysis revealed the expression of several virulence-associated proteins such as adhesins, invasins, and secretory and chemotaxis proteins that might be involved in various processes of attachment and invasion and as effectors during pathogenesis in the host. Further, the interaction of bacteria with the host cell (macrophages) was a major factor in the differential expression of these proteins. Finally, eight common differentially expressed RNA-protein pairs, predicted as virulent, outer membrane/extracellular proteins were validated by quantitative PCR. This is the first report using integrated multi-omics approach to identify critical factors involved in pathogenesis. Validation of these critical factors may lead to the identification of target antigens for the development of improved diagnostics and vaccines against leptospirosis. Leptospirosis is a zoonotic disease of global importance. It is caused by a Gram-negative bacterial spirochete of the genus . The current challenge is to detect the infection at early stage for treatment or to develop potent vaccines that can induce cross-protection against various pathogenic serovars. Understanding host-pathogen interactions is important to identify the critical factors involved in pathogenesis and host defense for developing improved vaccines and diagnostics. Utilizing an integrated multi-omics approach, our study provides important insight into the interaction of with human macrophages and identifies a few critical factors (such as virulence-associated proteins) involved in pathogenesis. These factors can be exploited for the development of novel tools for the detection, treatment, or prevention of leptospirosis.