A high amount of zeaxanthin, lutein, and meso-zeaxanthin are stored in the human macula, and they are associated with the risk reduction of human eye diseases such as age-related macular degeneration, glaucoma, and cataracts. The xanthophyll zeaxanthin and lutein usually coexist naturally and can be consumed from various fruits and vegetables. Zeaxanthin can be found in yellow-orange color fruits and vegetables like yellow corn, papaya, peaches, carrots, and mandarin oranges. Spirulina is also a microscopic and filamentous cyanobacterium containing a high concentration of zeaxanthin. Currently, zeaxanthin has been mainly produced by extraction and isolation from plants, but it is high-cost and energy-consuming. Therefore, the biosynthesis of zeaxanthin by microorganisms has been studied and developed recently. Many studies demonstrate that bacteria and microalgae are the most common naturally occurring zeaxanthin-accumulating microorganisms. Escherichia coli or yeast are broadly reported as engineered microorganisms for zeaxanthin production by regulating the biosynthetic pathway and the overexpression of the constructed gene. This chapter will discuss biosynthetic pathways of zeaxanthin, zeaxanthin production by natural zeaxanthin-accumulating method, and metabolic engineered microorganisms. At the end of the chapter, perspectives concerning the innovative strategies for further developing zeaxanthin production and its trends to improve the metabolically engineering microorganisms will be discussed.