The objective of the paper was to study the anti-tumor effect of total glycosides from Radix paeoniae rubra in S180 tumor-bearing mice, and to preliminarily explore its mechanism of action. Mice were made into S180 solid tumor model, grouped and administered with the extracts; tumor inhibition rate was measured by harvesting the tumors, and serum IL-2 and IL-4 levels were measured by taking blood samples. Total glycosides of Radix paeoniae rubra significantly inhibited the growth of tumor cells in tumor-bearing organisms, enhanced the cytotoxic activity of NK cells, and increased the serum IL-2 and IL-4 levels. Total glycosides of Radix paeoniae rubra have some anti-tumor effect in vivo, which might have been accomplished through the regulation of the immune system.