Lignosulfonic acid is a waste lignin produced from the sulfite pulping of softwood. We investigated the effect of lignosulfonic acid on α-glucosidase and found that lignosulfonic acid produced a reversible and non-competitive inhibition of the enzyme activity. Moreover, in human colorectal adenocarcinoma cells, lignosulfonic acid inhibited 2-deoxyglucose uptake, while in vivo studies demonstrated a significant reduction in the blood glycemic response to sucrose or glucose ingestion in rats treated with lignosulfonic acid. Feces of rats fed a diet supplemented with 5% lignosulfonic acid had higher sugar content compared to those of rats fed a control diet. These results suggest that lignosulfonic acid suppresses the rise in blood glucose levels through inhibition of α-glucosidase activity and intestinal glucose absorption. Additionally, lignosulfonic acid suppressed increase of blood glucose level in diabetic KK-Ay mice. These results suggest that lignosulfonic acid may be useful as an anti-diabetes agent.