Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA ( Cg ) (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA (Cg) was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA(Cg). The pobA (Cg) is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.