D-tagatose is a rare, naturally occurring low-calorie hexose, with a sweetness of 92% sucrose but only 1/3 of the calories. It has beneficial functions in lowering blood sugar, controlling obesity, preventing dental caries, and improving intestinal flora. In recent years, biotechnological routes to D-tagatose production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of the properties and applications of D-tagatose, with a focus on the current developments in the production of D-tagatose using enzymatic transformation and whole-cell catalytic synthesis. The biosynthetic pathways and the types and characteristics of the catalytic enzymes involved have been summarized, providing a reference for the design of D-tagatose synthesis pathways. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial D-tagatose production in the future.