Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic progressive lung disease that is increasing in incidence. Host genetic factors are associated with NTM-PD susceptibility. However, the heritability of NTM-PD is not well understood. Here, we perform a two-stage genome-wide association study (GWAS) and discover a susceptibility locus at 16p21 associated with NTM-PD, especially with pulmonary Mycobacterium avium complex (MAC) disease. As the lead variant, rs194800 C allele augments protein kinase C beta (PRKCB) gene expression and associates with severer NTM-PD. The functional studies show that PRKCB exacerbates M. avium infection and promotes intracellular survival of M. avium in macrophages by inhibiting phagosomal acidification. Mechanistically, PRKCB interacts with subunit G of the vacuolar-H