The aim of this study was to evaluate the permeation properties of gentamicin (G) in a novel dry powder form for inhalation through an artificial mucus model. Moreover, since respiratory infections sustained by Pseudomonas are a major cause of sickness and death in CF patients, the susceptibility of P. aeruginosa to engineered G powders was investigated. Micronized G and G/leucine (85:15) formulations were produced by co-spray-drying, using process parameters and conditions previously set. Powders were characterized in terms of yield, drug content and aerodynamic profiles, analyzed by Andersen Cascade Impactor. Different mucus models were prepared, showing composition and viscosity similar to those of the native CF mucus. To investigate the impact on drug permeation, Franz-type vertical diffusion cells were used; the powders were applied directly on a synthetic membrane with or without the interposition of the artificial mucus layer. In buffer, gentamicin showed a diffusion controlled release; the presence of leucine reduced powder wettability and, consequently, the permeation rate. Otherwise, mucus delayed drug permeation from both G and G/leucine formulations, with a faint influence of the aminoacid. Antimicrobial tests revealed that G/leu engineered particles are able to preserve the antipseudomonal activity, even in presence of the mucus.