Sugarcane leaves have been extensively consumed as a herbal tea for centuries in China, which is an economically important crop containing multiple health benefits. Here we report the structure and in vitro antitumor activity of a purified sugarcane leaf polysaccharide (SLP70–1). SLP70–1 is a homogeneous heteropolysaccharide made up of mannose, glucose, and galactose with a molar ratio of 1.00: 1.89: 2.83. Additionally, the molecular weight of SLP70–1 was 3.23 kDa. Linkage together with NMR examination demonstrated the structural foundation of SLP70–1 were consisted of →4)-α-D-Galp-(1→, →4)-α-D-Glcp-(1→, →2)-β-D-Manp-(1→, →3)-β-D-Galp-(1→, →4,6)-β-D-Galp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Glcp-(1→ and α-D-Glcp-(1→,and the side chain was confirmed to be →4)-α-D-Glcp-(1→ and →6)-α-D-Galp-(1→. According to an in vitro cell experiment, SLP70–1 could markedly decrease the survival ability of cancer cells exhibiting concentration dependence, including the survival rate of cancer cells in the lungs, colon, cervix, kidney, and breast, implying potent antitumor activity. In summary, SLP70–1 exhibits features that qualify it as a promising antitumor adjuvant candidate.