miR-21 has been shown to regulate multiple mRNAs and cause tumor progression and metastasis. However, whether miR-21-mediated posttranscriptional regulation is involved in antigen presentation and anti-mycobacterial responses remains unclear. Here, we report that miR-21 can be induced after Bacillus Calmette-Guerin (BCG) vaccination by NF-kB activation. miR-21 suppressed IL-12 production by targeting IL-12p35, which impaired anti-mycobacterial T cell responses both in vitro and in vivo. Additionally, miR-21 also promoted dendritic cell (DC) apoptosis by targeting Bcl-2. Therefore, miR-21 may potentially be involved in fine-tuning of the anti-mycobacterial Th1 response and in regulating the efficacy of BCG vaccination.