In order to understand sugar metabolism in stalk of the sweet sorghum, content of glucose, fructose and sucrose and the activities of neutral invertase (NI), soluble acid invertase (SAI), sucrose phosphate synthase (SPS) and sucrose synthase (SS) from six internodes of Cowley, Ramada and MN-2747 sweet sorghum (Sorghum bicolor (L.)Moench) cultivars were assayed. The results shown that content of fructose, glucose and sucrose were different significantly within three cultivars, were 2.32~4.34mg/g, 2.30~4.14mg/g and 35.92~95.92mg/g respectively. Content of fructose and glucose showed a "U" type trend and content of sucrose had no significant changes, only a slight increase, with the internodes number (1~11). Enzyme activities of NI, SAI, SPS and SS in mature stem are generally low, and showed a decreasing trend with the internodes number (1~11). It was negatively correlated between sucrose content and SAI activity (R=-0.71), and no relationship with NI, SPS and SS activity in individual internodes. SAI is a key enzyme controlling sugar metabolism in the sweet sorghum stalk.