The structural evolution of nanostructured Nb-doped magnesium film samples and its correlation with the change of the H2 desorption kinetics after successive H2 sorption cycles at 623 K was investigated by different techniques. The variation of the dispersed Nb fraction and the Nb clusterization was followed by extended x-ray absorption fine structure (EXAFS), while the progressive Mg nanostructuring was monitored by x-ray diffraction. The presence of vacancylike defects and their evolution was studied using positron annihilation lifetime spectroscopy and Doppler broadening spectroscopies. It was found that, with successive H2 sorption cycles: (i) the H2 desorption kinetics progressively becomes slower until stationary conditions are reached and (ii) the Nb dopant atoms, dispersed in the nanocrystalline Mg layers, aggregate, forming nanoclusters. Our results show that the progressive Nb aggregation drives the H2 desorption kinetics. EXAFS analysis show that fast desorption kinetics is due to the presence of small (∼1 nm) Nb aggregates rather than Nb atoms dispersed into the Mg matrix. With cycling, the Nb aggregates progressively grow, forming larger bcc Nb nanoclusters and the H2 desorption kinetics becomes slower. In the as-deposited Nb-doped Mg samples, analysis of the positron data reveals the presence of intragranular vacancylike defects and of vacancy clusters which are inferred to be mainly located at the grain boundaries of the nanocrystalline Mg layers. With H2 cycling: (i) a decrease of the atomic fraction of the intragranular vacancylike defects after the first two sorption cycles was observed, and (ii) an increase of the atomic fraction of vacancy clusters at grain boundaries and the appearance of vacancylike defects located at the interface between the Nb aggregates and the Mg matrix was probed. It was also found that the kinetics follows a nucleation and growth mechanism and, under stationary conditions, the Mg nucleation is controlled by vacancy-decorated bcc Nb nanoclusters rather than by vacancy clusters, as in undoped Mg samples.