A new congener of chuangxinmycin (CM) was identified from <i>Actinoplanes tsinanensis</i> CPCC 200056. Its structure was determined as 3-methylchuangxinmycin (MCM) by 1D and 2D NMR. MCM could be generated <i>in vivo</i> from CM by heterologous expression of the vitamin B<sub>12</sub>-dependent radical SAM enzyme CxnA/A<sub>1</sub> responsible for methylation of 3-demethylchuangxinmycin (DCM) in CM biosynthesis, indicating that CxnA/A<sub>1</sub> could perform iterative methylation for MCM production. <i>In vitro</i> assays revealed significant activities of CM, DCM, and MCM against <i>Mycobacterium tuberculosis</i> H37Rv and clinically isolated isoniazid/rifampin-resistant <i>M. tuberculosis</i>, suggesting that CM and its derivatives may have potential for antituberculosis drug development.