The interplay between protein dynamics and catalysis remains a fundamental question in enzymology. We here investigate the ns-timescale dynamics of a light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), a photoenzyme crucial for chlorophyll synthesis. LPORs catalyze the light-triggered trans addition of a hydride and a proton across the C17=C18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). Because of the lack of an LPOR structure, the global structural and dynamic consequences of LPOR/Pchlide/NADPH ternary complex formation remain elusive. Moreover, photoactivation of LPORs by low-light preillumination is controversially discussed as unequivocal proof for this phenomenon is lacking. By employing quasielastic neutron spectroscopy (QENS), we show that the formation of the ternary holoprotein complex as well as photoactivation lead to progressive rigidification of the protein. These findings are supported by thermostability measurements, which reveal different melting behavior and thermostabilities for the apo- and holoprotein ternary complexes. Molecular dynamics simulations in good agreement with the experimental QENS results suggest that the increased flexibility observed for the apoprotein stems from structural fluctuations of the NADPH and Pchlide substrate binding sites of the enzyme. On the basis of our results, in conjunction with activity and stability measurements, we provide independent proof for LPOR photoactivation, defined as a process that modifies the protein structure and dynamics, resulting in an increased substrate turnover. Our findings advance the structural and dynamic understanding of LPORs and provide a first link between protein dynamics and catalysis for this enzyme class.