The whiA gene is widely distributed among Gram-positive bacteria. Although the encoded protein has conserved N-terminal homing endonuclease scaffold and C-terminal helix-turn-helix DNA-binding domains, whiA plays a unique physiological role in its host organisms, reflecting a long history of evolution. Here, we used genetic approaches to unveil the physiological function of whiA in Corynebacterium glutamicum. We found that cells lacking whiA (Delta whiA) were unable to grow in minimal medium containing glucose, although reduced growth was observed in complex medium. The Delta whiA strain showed altered transcription of the cell division genes ftsZ, sepF, ftsK, crgA, divIVA, and amiC genes. Accordingly, Delta whiA cells exhibited large, elongated, branched, and bud-shaped morphologies. In addition, some genes, including fas-IA, fas-IB, accD1, and cmrA, which help synthesize the fatty acid and cell envelope component mycolic acid, showed altered transcription in the Delta whiA strain. Further, treS, treY, otsA, and otsB, which are involved in the biosynthesis of the outer envelope component trehalose, were down-regulated in the Delta whiA strain. 2D-PAGE analysis of the Delta whiA mutant showed that proteins involved in other cellular activities were also affected by the loss of whiA. These findings suggest that C. glutamicum whiA plays a critical role in cell division, envelope formation, and general cell physiology.