Due to the natural resistance of non-tuberculous mycobacteria(NTM) to lots of antibiotics,treatment of diseases caused by NTM is often long-termed but less successful.The main goal of this study was to evaluate the in vitro susceptibility to clofazimine of 209 isolates consisting of different NTM species isolated in Beijing,China. Furthermore,47 reference strains were also tested, including 30 rapidly growing mycobacterium (RGM) species and 17 slowly growing mycobacterium(SGM) species.The potential molecular mechanism contributing to clofazimine resistance of NTM was investigated as well.Clofazimine exhibited excellent activities against both reference strains and clinical isolates of different SGM species,and most of the strains had MICs far below 1μg/ml.Although the majority clinical isolates of M. abscessus and M. fortuitum had MICs higher than 2μg/ml, 17 out of the 30reference strains of different RGM species had MICs below 1μg/ml in vitroAccording tothe MIC distributions, the tentative epidemiological cut-off (ECOFF)values for M. kansassii,M. aviumand M. intracellulare were defined at 0.5μg/ml,1μg/ml and 2μg/ml, respectively. Intriguingly, single directional cross-resistance between bedaquiline and Cfz resistant isolates was observed among the tested NTM species.This study demonstrate that clofazimine had strong activities against most SGM species in vitro, and some RGM species as well.The data provided important insights on the possible clinical application of Cfz to treat NTM infections.