O3 is not only greenhouse gas but also a primary gaseous contaminant in the atmosphere. It has long-lasting effects on crop growth, yield and quality, and brings a series of ecological and environmental problems. A free-air controlled enrichment (FACE) system was applied to study the effect of elevated ozone concentration on activities of key enzymes of starch synthesis of Yangmai 16 in 2009-2010. The main-plot treatment had two levels of O3: ambient level (A-O3) and 50% higher than ambient level (E-O3). The main results were that accumulation rate of amylose, amylopectin and starch were represented in a single peak curve, and their content and accumulation amount rose gradually. The O3 elevation decreased the accumulation rate of amylose, amylopectin and starch amylase, reduced the accumulation amount of amylopectin and starch, and decreased the content of amylopectin and starch, but increased the content of amylose. With the increase of O3 concentration, the enzyme activity of grain granule-bound starch synthase (GBSS), soluble starch synthase (SSS) and starch branching enzyme (SBE) decreased after anthesis. The activities of GBSS and SSS had highly significant correlations with amylose, amylopectin and starch accumulation rate, and the activity of SBE had significant correlations with these items. So the O3 elevation decreased the activity of key enzymes of starch synthesis, which led to the variation of starch synthesis.