Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. Out of twenty cytochrome P450 enzymes encoded by the Mtb genome, cytochrome P450 121A1 (CYP121A1) is essential, and therefore remains a target of drug design efforts. CYP121A1 mediates a phenol coupling reaction of the tyrosine dipeptide cyclo-L-Tyr-L-Tyr (cYY). In this work, a structure and function investigation of dimerization was performed as an overlooked feature of CYP121A1 function. This investigation showed that CYP121A1 dimers form via intermolecular contacts on the distal surface and are mediated by a network of solvent-exposed hydrophobic residues. Dimerization of CYP121A1 contributes to specific substrate binding in vitro, with 75% loss of function when dimers are disrupted. 19F labeling and nuclear magnetic resonance of the enzyme FG-loop was also combined with protein docking to develop a working model of a functional CYP121A1 dimer. Preliminary crystallographic data of a CYP121A1 homodimer confirm the distal-to-distal orientation of the dimers. These findings suggest a novel role for CYP dimer formation as a contributing factor in determining substrate selectivity, while also providing important mechanistic insight regarding a relevant drug target in the development of novel anti-tuberculosis agents.