The aim of this research is to evaluate the intestinal permeation of a new formulation (NF) for the anti-retroviral didanosine (ddI) drug, using the everted gut sac model. The NF is composed by granules containing ddI incorporated in chitosan microspheres, plus free chitosan as an excipient. The permeation was evaluated across the three intestinal segments of adult male Wistar rats. The performance of ddI permeation from the NF was compared to the same granules without free chitosan and to buffered ddI tablets as control. The permeations across duodenum were higher than across jejune and ileum. The ddI from NF presented higher permeation and a crescent-shaped profile in duodenum compared to the other formulations. Such effects are provided by the superior mucoadhesiveness to the intestinal membrane and potentialize sustained release properties for NF. These results lead one to consider the novel formulation to be promising for ddI administration by oral route.