The antitumor activity of Lycium barbarum polysaccharide (LBP) has been reported, but the structure-bioactivity relationship has still not been fully elucidated. In this study, four water-soluble LBP fractions with serial different molecular weights (MWs) were separated from LBP, designated LBP-2, LBP-3, LBP-4, and LBP-5. After a characteristic analysis, the relationship between MW and antitumor activity of LBP was investigated both in vitro using murine hepatoma H22 cells and in vivo using H22 tumor-bearing mice. In vitro, the results showed that all the LBP fractions had significant inhibition on H22 cells, in which LBP-3 had the best activity. LBP-3 could induce apoptosis, mitochondrial membrane potential destruction, and S phase arrest in H22 cells. In vivo, the results showed that LBP-2, LBP-3, LBP-4, and LBP-5 could inhibit the tumor growth in H22 tumor-bearing mice by 18.18%, 37.97%, 9.09%, and 14.44%, respectively. However, only LBP-3 was able to decrease the tumor weight significantly in H22 tumor-bearing mice. Meanwhile, all the LBP fractions did not show significant toxicity to murine splenocytes, thymus, and spleen. Taken together, these results demonstrated that the antitumor activity of LBP was closely related to its MW, and LBP-3 with medium MW (40-350 kDa) was the main active fraction.