In order to investigate nitrogen fixation and accumulation in sugarcane under nitrogen supply, a barrel gravel culture was conducted in Guangxi province, the southwest China in 2012. The sugarcane varieties B 8 and ROC 22 were employed as the plant materials, which were inoculated either with or without endogenous nitrogen fixation bacteria at two levels of nitrogen supply, that is 0 N and 1/2 N. The nitrogenase activity in the stalks and total nitrogen in the leaves were determined at different growth stages of sugarcane. The results showed that the nitrogenase activity in stalks reached its peak at booming stage. The treatment with 1/2 N nitrogen supply significantly increased the nitrogenase activity in stalks and total nitrogen content in leaves compared that with 0 N for both with and without inoculation. The nitrogenase activity was more sensitive in B 8 than in ROC 22. ANOVA results revealed that the nitrogen supply, variety and inoculation had significant interaction effects on nitrogenase activity and total nitrogen content in sugarcane. Therefore, it was concluded that inoculation of endogenous nitrogen fixation bacteria and fertilization of a low amount of nitrogen were beneficial for the biological nitrogen fixation, cane yield so saving chemical fertilizer in sugarcane production.