Hybrid meta-GGA density functional theory MPWB1K functional is used to study the hydroxylation and ring-opening mechanism of 2-methyl-3-hydroxypyridine- 5-carboxylic acid oxygenase (MHPCO). This enzyme catalyzes the conversion of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) to α-(N-acetylamino- methylene) succinic acid (AAMS), which is the essential ring-opening step in the bacterial degradation of Vitamin B6. MHPCO belongs to the flavin-containing aromatic hydroxylases. However, MHPCO is capable of catalyzing a subsequent aromatic ring cleavage reaction to yield acyclic products rather than hydroxylated aromatic ones. Our calculations show that the rearomatization of the hydroxylated intermediate occurs spontaneously in aqueous solution, implying that the ring-opening process occurs inside the enzyme active site with limited water around. The instability of the hydroxylated intermediate of MHPCO is the main reason that the acyclic products are formed. Previously proposed mechanisms for the ring-opening step are studied, and are shown to be less likely to occur (ΔΔG≠298 >35 kcal/mol). Two new pathways with reasonable barrier heights (ΔΔG≠298 <15 kcal/mol) are reported herein, which are in accordance with all experimental information present to date.