3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are key enzymes catalyzing exergonic reactions of the formaldehyde-fixing reaction and the isomerization of sugar phosphate in the ribulose monophosphate (RuMP) pathway. This pathway, which was originally found in methylotrophic bacteria, is now recognized to be widespread in prokaryotes and has been shown to be involved not only in formaldehyde fixation and detoxification but also in pentose phosphate biosynthesis. In this review, we describe the genomic organization and regulation of the genes of the RuMP pathway and then discuss the physiological roles of this pathway in prokaryotes. We further describe the biochemical properties of HPS and PHI. Heterologous expression of HPS and PHI in various organisms allows them to metabolize and detoxify formaldehyde, and we also review recent progress in such applications in biotechnology.