Zinc protoporphyrin IX (ZnPPIX) has been considered as a promising red colorant for food industries as well as an anticancer drug. However, bio-based production of ZnPPIX from a renewable carbon source has not been reported yet. In this study, a fermentation process of the metabolically engineered Escherichia coli HAEM7 strain was optimized for the high-level production of ZnPPIX. To repurpose the HAEM7 strain that was originally developed for the production of heme into a producer of ZnPPIX, the concentrations of iron and zinc in the culture medium were rebalanced. Next, the concentration of zinc in the feeding solution was optimized to improve ZnPPIX production. Moreover, the pH control strategy, induction point, and the strategy of increasing the cell density, which were optimized in the accompanying paper for the high-level production of heme, were applied together. In the optimized fermentation process, the HAEM7 strain produced 2.2 g/L ZnPPIX with a productivity of 39.9 mg/L/h. The fermentation process and strategies reported here will expedite establishing industry-level production of ZnPPIX.