Infectious diseases caused by intracellular bacteria represent a challenge in the antibiotic therapy. Pulmonary tuberculosis is characterized by the involvement of macrophages infected with a large number of Mycobacterium tuberculosis. The lichen metabolite Usnic Acid (UA) has been reported to offer remarkable antimycobacterial activity. Liposomes have long been recognized as good carriers in targeting of drugs to cells. In this framework, the aim of this study was to assess the antimicrobial activity of UA-loaded liposomes against M. tuberculosis and to investigate their interactions with macrophages. Liposomes were prepared using the hydration lipid film technique with subsequent sonication. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against M. tuberculosis H37Rv were determined according to the microdilution Alamar blue assay. The cytotoxicity of UA was evaluated on J774 macrophages and the cell viability was detected by the standard MTT assay. The cellular uptake studies were carried out on J774 macrophages using fluorescence spectroscopy. The values of MIC and MBC for free UA were 8 and 32 mg/ml, respectively. In contrast, UA-loaded liposomes exhibited a MIC of 6 mg/ml and a MBC of 16 mg/ml. The concentration required to inhibit 50% of cell proliferation (IC50) were 20 and 9.5 mg/ml for free and encapsulated UA, respectively. The cytotoxic effect of UA-loaded liposomes on J774 macrophages was thus two-fold higher than free UA. An enhancement in the intracellular uptake of UA-loaded liposomes was verified by the highest detected fluorescence emission (21.57 x 104  c.p.s) in comparison with free UA (95.44 x 103 c.p.s). Besides, the liposomal formulation provided an almost invariable amount of UA inside macrophages during a long time (14 h). These results indicated that a strongly interaction exists between liposomes and J774 macrophages, facilitating UA penetration into cells and thereby remarkably improving its activity against the M. tuberculosis. Supported by: CNPq/MCT, CAPES, Rede de Pesquisa em Tuberculose.