The protocatechuate (PCA) 4,5-cleavage pathway is one of the key catabolic routes for the degradation of various aromatic compounds, such as lignin-derived low-molecular-weight aromatic compounds, phthalate isomers, and fluorene. In this pathway, the aromatic ring of PCA is initially cleaved by PCA 4,5-dioxygenase, and the resultant product is degraded to pyruvate and oxaloacetate, via the five-enzymatic steps. The gene organization of the PCA 4,5-cleavage pathway genes is divided into two types, which constitute several transcriptional units (Sphingobium type) or a single operon (Comamonas type). This chapter summarizes properties of each enzyme involved in this pathway and focuses on the recently characterized transcriptional regulation of the pathway genes. We also described a newly found enzyme involved in the pathway, 4-oxalomesaconate tautomerase. The role of the PCA 4,5-cleavage pathway in the catabolism of lignin-derived aromatic compounds is discussed at the end of this chapter.