Abstract: Sesterterpenoids, composed of five isoprene units and biosynthetically derived from geranylfarnesyl diphosphate (GFDP), are a class of the precious terpenoids with approximately 1 300 natural products known to date. Natural sesterterpenoids are widely distributed and possess extreme structural complexity and diversity and remarkable biological activity. In recent decades, a series of important progresses have been made in sesterterpenoid biosynthesis with the development of genome mining and heterogeneous expression technologies. This paper mainly focuses on the advances in sesterterpenoid biosynthesis, including the biochemical functions and catalytic mechanisms of GFDP synthases, sesterterpene synthases and oxidases. This review would lay the foundation for in-depth investigation on the biosynthesis, biological activities and synthetic biology of sesterterpenoids.