During the course of infection, pathogenic mycobacteria including Mycobacterium tuberculosis (M. tb) encounter host environments of variable oxygen tension, ranging from the hypoxic center of granulomas to the most oxygenated region in the lung cavities. Mycobacterial responses to changes of oxygen tension are critically related to infection outcomes, such as latency and reactivation. WhiB4 is an iron-sulfur containing transcription factor that is highly sensitive to oxygen exposure. In this study, we found that WhiB4 of Mycobacterium marinum (M. marinum), a pathogenic mycobacterial species that is closely related to M. tb, is required for its virulence. M. marinum ΔwhiB4 exhibited defective intracellular replication in macrophages and diminished virulence in zebrafish. Histology analysis revealed that the host had successfully controlled ΔwhiB4 bacteria, forming well-organized granulomas. RNA-seq analysis identified a large number of pe/ppe genes that were regulated by WhiB4, which provides an explanation for the essential role of WhiB4 in M. marinum virulence. Several antioxidant enzymes were also upregulated in ΔwhiB4, supporting its role in modulation of oxidative stress response. Taken together, we have provided new insight into and proposed a model to explain the physiological role of WhiB4.