As a health-beneficial fruit, watermelon is widely consumed by people around the world. However, components responsible for the health benefits are not yet determined. As watermelon contains a large amount of polysaccharides, these carbohydrates might play an important role in the health benefits. In this work, polysaccharide from watermelon rinds (PWR) was extracted by papain digestion, purified and characterized by GC–MS, SEC/MALS/VD/DRI, FTIR and 1D and 2D NMR which revealed the glycosidic linkages, their locations in branches and backbone. The monosaccharide composition revealed that the extracted polysaccharide was composed of galactose (38.26%), arabinose (26.12%), rhamnose (17.86%), mannose (9.94%), xylose (5.10%) and glucose (2.70%) with a percentage of uronic acid of 45%. A combination of CPG and NMR analysis showed that the extracted polysaccharide is arabinogalactan linked to type I rhamnogalacturonan. we notice that the arabinogalactan was formed by →6)-β-D-Galp-(1→ as backbone with short branching of arabinose linked in α 1 → 3, rhamnose linked in α 1 → 4, mannose linked in β 1 → 6 and galactose branches linked in β 1 → 3. Furthermore, PWR exhibited obvious cytotoxicity ability to human laryngeal carcinoma Hep-2 cells in a dose-and time-dependant manner.