Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In humans, point mutations on PPO are responsible for the dominantly inherited disorder disease variegate porphyria (VP). It is found that several VP-causing mutation sites are located on an α-helix cluster (consisting of α-5, α-6, and α-7 helix, named the G169 helix cluster) of human PPO, although these mutation sites are outside the active site of the human PPO. In this work, we investigated the role of the G169 helix cluster via site-directed mutagenesis, enzymatic kinetics, and computational studies. Kinetic studies showed that mutations on the G169 helix cluster affect the activity of PPO. The MD simulation showed that mutations on the G169 helix cluster reduced the activity of PPO by affecting the proper orientation of substrate protoporphyrinogen within the active site of PPO and possibly the dipole moment of the G169 helix cluster. Moreover, the mutation abolished the interaction between the mutated site and other residues, thus affecting the secondary structure and hydrogen bond interactions within the G169 helix cluster. These results indicated that the integrity of the G169 helix cluster is important for the stabilization of protoporphyrinogen within the active site of PPO to facilitate the interaction between protoporphyrinogen and cofactor FAD and provide a proper electrostatic environment for the activity of PPO. Our result provides new insight into understanding the relationship between the structure and function of PPO.