Pemetrexed (PMX) is a multi-targeted antifolate drug used for the treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer. Hyaluronan (HA) in blood is well known as a disease marker of MPM. We synthesized PMX-conjugated hyaluronan (HA-ADH-PMX) for the first time to develop a novel anticancer chemotherapeutic agent. HAs with different molecular weights (76 and 130 kDa) were first derivatized with adipic dihydrazide (ADH) and then conjugated to PMX. The obtained HA-ADH-PMX retained inhibitory activity against folate metabolism enzymes; thymidylate synthase was inhibited to the same extent as native PMX, whereas the inhibition constant against dihydrofolate reductase was 3.3% for 76 kDa HA-ADH-PMX and 12% for 130 kDa HA-ADH-PMX when compared with that of native PMX. The in vitro cytotoxicity of HA-ADH-PMX from both molecular weights against MPM cell lines was lower than that of native PMX. On the other hand, intrapleural administration of 76 kDa HA-ADH-PMX resulted in a survival rate of MPM model mice comparable to that with native PMX, suggesting the potential for future MPM therapy.