Abstract Non-Tuberculous Mycobacteria (NTM) are ubiquitous in nature, present in soil and water, and cause primary and disseminated infections in the immune-compromised individuals. NTM infections are surging in recent years at 8% annually, due to an increase in immune-suppressed population, medical interventions, and underlying lung diseases. The innate immune responses, frontiers for controlling infections and dissemination, are poorly defined during NTM infections. Here, we define the role of CBLB, an E3-ubiquitin ligase, for innate immune responses and disease progression in a mouse model of NTM infection in the absence of antigen-specific T-cell responses. We found that NTM was readily disseminated into the tissues in the Cblb KO mice in a time- and infection route-dependent manner, compared with the WT group. We uncovered defects in many innate immune cells in Cblb KO mice: poor responses with NK cells, inflammatory monocytes, neutrophils, and activation of conventional dendritic cell responses. Strikingly, Cblb-deficient macrophages were able to produce higher ROS in both in vivo & in vitro compared with the WT group. Histopathological studies buttressed the role of Cblb for disseminated NTM infection. Collectively, Cblb plays a dichotomous role in innate immunity, and may help prevent the dissemination of NTM in the infected host. Our studies illuminate the translational implications for controlling NTM infections by modulating the innate immune immunity.