The usnic acid (UA), a biosynthetic product from several epiphytic lichens, has been reported as a promising chemotherapeutic agent for human tuberculosis. The potential of controlled release in optimizing drug targeting has offered a tremendous advancement in the manipulation of novel dosage forms such as nanocapsules. In this way, the aim of this study is to report the antimycobacterial activity of the usnic acid from Cladonia substelatta encapsulated into nanocapsules (NC-UA) and evaluated its cellular uptake. The NC-UA was prepared using the method of interfacial deposition of a pre-formed polymer. The size distribution and zeta potential of particles were measured using a ZetasizerÒ. The antimycobacterial activity of UA and NC-UA was determined on M. tuberculosis H37Rv (ATCC 27294) using minimum inhibitory concentration (MIC). Cellular uptake of NC-UA was investigated on J774 macrophages using fluorescence spectroscopy through the detection of the UA fluorescence emission. NC-UA presented 274 ± 74 nm mean diameter of particles with a polydispersity index of 0.3, which indicated a narrow particle size distribution. The NC-UA exhibited a zeta potential value of -28 ± 8 mV. The MIC of UA and NC-UA were 25 mmol.L-1 and 12 mmol.L-1, respectively. Thus the nanoencapsulation increased two-fold the antimycobacterial activity of UA. The highest emission of fluorescence (95.44 x 103 c.p.s) occurred at 10 hours after cellular exposition to NC-UA followed by a gradual decreased until 72 hours (62.4 x 103 c.p.s). UA exhibited a maximum of fluorescence at 8 hours. This behavior is expected considering the burst release of the UA from the nanoparticles. The observed slow release of UA occurring even after 72 hours, clearly indicate a sustained release.  In conclusion, the nanoencapsulation of UA allowed an improvement on its in vitro antimycobacterial effectiveness and induced the cellular uptake. Supported by: CNPq/MCT, Rede de Pesquisa em Tuberculose.