The enhanced permeability and retention (EPR) effect is a unique pathophysiological phenomenon of solid tumors that sees biocompatible macromolecules (>40 kDa) accumulate selectively in the tumor. Various factors have been implicated in this effect. Herein, we report that heme oxygenase‐1 (HO‐1; also known as heat shock protein 32) significantly increases vascular permeability and thus macromolecular drug accumulation in tumors. Intradermal injection of recombinant HO‐1 in mice, followed by i.v. administration of a macromolecular Evans blue–albumin complex, resulted in dose‐dependent extravasation of Evans blue–albumin at the HO‐1 injection site. Almost no extravasation was detected when inactivated HO‐1 or a carbon monoxide (CO) scavenger was injected instead. Because HO‐1 generates CO, these data imply that CO plays a key role in vascular leakage. This is supported by results obtained after intratumoral administration of a CO‐releasing agent (tricarbonyldichlororuthenium(II) dimer) in the same experimental setting, specifically dose‐dependent increases in vascular permeability plus augmented tumor blood flow. In addition, induction of HO‐1 in tumors by the water‐soluble macromolecular HO‐1 inducer pegylated hemin significantly increased tumor blood flow and Evans blue–albumin accumulation in tumors. These findings suggest that HO‐1 and/or CO are important mediators of the EPR effect. Thus, anticancer chemotherapy using macromolecular drugs may be improved by combination with an HO‐1 inducer, such as pegylated hemin, via an enhanced EPR effect. (Cancer Sci 2012; 103: 535–541)