To obtain insight into the motional features of proteins for enzymatic function, we studied binding reactions between ferredoxin-NADP+ reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based magnetic relaxation and hydrogen/deuterium exchange (HDex). Fd–FNR binding was accompanied by endothermic reactions and driven by the entropy gain. Component-wise analysis of the net entropy change revealed that increases in the conformational entropy of the Fd–FNR complex contributed largely to stabilizing the complex. Intriguingly, analyses of magnetic relaxation and HDex rates with X-ray B factor implied that Fd binding led to both structural stiffening and softening of FNR. Enhanced FNR backbone fluctuations suggest favorable contributions to the net conformational entropy. Fd-bound FNR further showed that relatively large-scale motions of the C terminus, a gatekeeper for interactions of NADP+(H), were quenched in the closed form, thereby facilitating exit of NADP+(H). This can provide a first dynamic structure-based explanation for the negative cooperativity between Fd and NADP+(H) via FNR.