Films of poly(3-hydroxybutyrate) (PHB) were crystallized from the melt by different thermal treatments and submitted to enzymatic degradation by using a PHB depolymerase purified from Aureobacterium saperdae culture. The morphology and the supermolecular structure of PHB films were investigated to explain differences in the kinetics of enzymatic degradation. Differential scanning calorimetry, optical microscopy, wide-angle X-ray diffraction and small-angle X-ray scattering were employed to characterize the PHB films. A decreasing of enzymatic degradation rate was observed with the increasing of crystallinity and crystal dimension of the PHB films. PHB samples showing the same degree of crystallinity and similar value of lamellar thickness were prepared using different isothermal crystallization and annealing temperatures. The differences in the enzymatic degradation rate of these films were explained in terms of morphological parameters.