Host defense against intracellular pathogens depends upon innate and adaptive antimicrobial effector pathways. TLR2/1-activation of monocytes leads to the vitamin D-dependent production of cathelicidin and, at the same time, an antimicrobial activity against intracellular Mycobacterium tuberculosis. To determine whether induction of cathelicidin was required for the vitamin D-triggered antimicrobial activity, the human monocytic cell line THP-1 was infected with M. tuberculosis H37Ra and then activated with the active vitamin D hormone 1, 25-dihydroxyvitamin D 3 (1, 25D 3). 1, 25D 3 stimulation resulted in antimicrobial activity against intracellular M. tuberculosis and expression of cathelicidin mRNA and protein. Using small interfering RNA (siRNA) specific for cathelicidin, 1, 25D 3-induced cathelicidin mRNA and protein expressions were efficiently knocked down, whereas a nonspecific siRNA control had little effect. Finally, 1, 25D 3-induced antimicrobial activity was completely inhibited in the presence of siRNA against cathelicidin, instead leading to enhanced intracellular growth of mycobacteria. These data demonstrate that cathelicidin is required for the 1, 25D 3-triggered antimicrobial activity against intracellular M. tuberculosis.