We derive the one-instanton effective action of N=4 super Yang-Mills theory in terms of the N=4 on-shell superfields. In the Coulomb branch, instantons correct both the MHV and next-to-next-MHV higher derivative terms D^4F^{2n+2} and F^{2n+4}. We confirm at the non-perturbative level the non-renormalization theorems for MHV F^{2n+2} terms that are expected to receive perturbative corrections only at n-loops. We compute also the one and two-loop corrections to the D^4F^4 term and show that its completion under SL(2,Z) duality is consistent with the one-instanton results.