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This work established a microwave-assisted procedure for polysaccharide extraction from Polygonatum odoratum. Response surface methodology was used to optimize microwave-assisted extraction parameters (extraction temperature, water-to-raw material ratio, microwave power, and extraction time) by implementing a three-level, fourvariable Box–Behnken experimental design in a single-factor investigation. Three-dimensional response surfaces were plotted by Design-Expert, and result indicated the absence of interaction effects of extraction temperature and microwave power. A polysaccharide yield of approximately 17.49% was obtained under the following optimized conditions: temperature of 57°C, microwave power of 300 W, extraction time of 10 min, and water-to-raw material ratio of 23:1. The results of preliminary in vitro antitumor activity tests showed that polysaccharides derived from P. odoratum could inhibit growth of A549 cells in a dose-dependent manner. Moreover, 4-400 μg/mL of these polysaccharides exerted no significant cytotoxicity to Panc-1 cells.