To investigate the influence of molecular weight ( M w) on the anti-inflammatory activity of β-D-glucan from Ganoderma lucidum, ultrasonic irradiation was applied to treat the β-D-glucan (GLP, 2.42 × 10 6 g/mol) solution to obtain two degraded fractions with molecular weight of 6.53 × 10 5 g/mol (GLPC) and 3.49 × 10 4 g/mol (GLPN). Structural analysis proved that the degraded fractions possessed similar repeated units with the original β-D-glucan. The in vitro anti-inflammatory activity studies showed that all fractions could significantly inhibit LPS-induced expression of cytokines including TNF-α, IL-8, MIF and MCP-1 in Caco-2 cells at certain concentrations. Moreover, GLPC and GLPN exhibited better anti-inflammatory activity than GLPC. The intestinal anti-inflammatory activity evaluated by dextran sulfate sodium (DSS)—induced colitis mice model showed that intragastric administration of GLPN (lower M w fraction) could significantly recover inflamed tissues of mice. Compared with GLP and GLPC, GLPN exhibited stronger ability to inhibit the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). The results revealed that M w of β-D-glucan influenced its anti-inflammatory activity and decreasing of M w would improve the activity, which provided evidence for the potential use of β-D-glucan from G. lucidum as anti-colitis ingredients.