Serine hydroxymethyltransferase (SHMT), a ubiquitous representative of the family of fold-type I, pyridoxal 5'-phosphate (PLP) dependent enzymes, catalyzes the reversible conversion of tetrahydrofolate (H4PteGlu) and serine to 5,10-CH2-H4PteGlu and glycine. Together with thymidylate synthase (TS) and dihydrofolate reductase (DHFR), SHMT participates to the thymidylate (dTMP) biosynthetic process. Elevated SHMT activity has been coupled to the increased demand for DNA synthesis in tumour cells. However, SHMT is the only enzyme of the thymidylate cycle yet to be targeted by chemotherapeutics. In this study, the interaction mode of SHMT with pemetrexed, an antifolate drug inhibiting several enzymes involved in folate-dependent biosynthetic pathways, was assessed. The mechanism of SHMT inhibition by pemetrexed was investigated in vitro using the human recombinant protein. The results of this study showed that pemetrexed competitively inhibits SHMT with respect to H4PteGlu with a measured Ki of 19.1±3.1 μM; this value was consistent with a Kd of 16.9±5.0 μM, measured by isothermal titration calorimetry. The binding mode of pemetrexed to SHMT was further investigated by molecular docking. The calculated interaction energy of pemetrexed in the active site of SHMT was -7.48 kcal/mol, and the corresponding predicted binding affinity was 36.3 μM, in good agreement with Kd and Ki values determined experimentally. The results thus provide insights into the mechanism of action of this antifolate drug and constitute the basis for the rational design of more selective inhibitors of SHMT.