This paper describes the mechanistic characterization of a yeast beta-1,3-D-glucan synthase (GS). GS is a proven target of FDA-approved antifungal drugs, but its catalytic mechanism is poorly understood. Using a newly developed size exclusion chromatography (SEC) assay, we were able to monitor the chain elongation for the first time and found that GS catalyzes the formation of a long glucan (~2,000 – 7,000 mer) at an amazing efficiency (~50-60 Glc unit polymerized per sec). The use of substrate analogs also revealed that the enzyme catalyzes the polymerization at the nonreducing end. The study also discusses the first comprehensive model of the GS catalytic cycle.