We synthesized positively charged biodegradable hydrogels from poly(propylene fumarate-co-ethylene glycol) block copolymer and agmatine-modified poly(ethylene glycol)-tethered fumarate by radical crosslinking, and investigated the effect of the guanidino group of agmatine on vascular smooth muscle cell adhesion and protein adsorption to the hydrogels. In the presence of serum, the number of adherent smooth muscle cells per unit surface area increased dose-dependently from 15 to 75% of the initial seeding density at 20 h as the initial agmatine-modified monomer content increased from 0 to 200 mg/g. Cell spreading also depended on the initial monomer content. In the absence of serum, the number of adherent cells per unit surface area increased slightly from 10 to 17% of the initial seeding density as the initial monomer content increased from 0 to 200 mg/g. Cell adhesion increased significantly by adding exogenous vitronectin to serum-free medium, whereas exogenous fibronectin addition did not enhance cell adhesion. The enzyme-linked immunosorbent assay of fibronectin and vitronectin adsorbed onto the hydrogels revealed that the incorporation of positive charges into the hydrogels enhanced vitronectin, but not fibronectin, adsorption significantly. These results suggest that the guanidino group of agmatine enhanced cell adhesion by promoting the adsorption of serum components, and vitronectin may be one of the components.