Dihydrofolate Reductase (DHFR) catalyzes the reduction of dihydrofolate (H2F) to tetrahydrofolate. On the basis of 10-12.5 ns molecular dynamics simulations of two conformations (closed and occluded) of the ternary DHFR/NADPH/H2F complex from Escherichia coli and a free energy perturbation approach, we have calculated the pKa value for the N5 atom in H2F. Our results suggest that the N5 atom in H2F is responsible for the pH dependency of the catalyzed reaction, meaning that DHFR facilitates protonation of H2F by approximately 4 pKa units. The mechanism behind this increase is due to favorable electrostatic interactions between the Asp27 residue and a proton at the N5 atom. The electrostatic interactions are enhanced by a hydrophobic active site, which to a large extent is made hydrophobic by the M20 loop in DHFR. Moreover, we find that the conformation imposed on H2F by DHFR to some extent also favors protonation of the N5 atom. Our results add support to previous findings and suggestions by Callender and co-workers [e.g., Deng, J.; Callender, R. J. Am. Chem. Soc. 1998, 120, 7730-7737] and explain why mutation of Asp27 may lead to severely reduced activity at neutral pH.