Pyruvate is an important metabolite in the central metabolism of living cells. It has been widely applied in food, pharmaceutical, and agrochemical industries. Pyruvate can be produced by both chemical and biological systems. Novel biotechnological systems that can yield pyruvate have been the focus of process development in pyruvate production. In this review, we summarize recent developments related to pyruvate production by biotechnological systems, with emphasis on the enzymatic synthesis of pyruvate from the cheaper substrate lactate.