Progressive accumulation of large intracellular vesicles is a hallmark of cell pathology in lysosomal storage diseases. Accumulating vesicles are commonly identified as crippled lysosomes engorged with incompletely digested materials resulting form the genetic defect. Our study of cortical neurons of the mouse model of mucopolysaccharidosis IIIB (Sanfilippo syndrome type B), a lysosomal storage disease with predominant neurological manifestations, showed efficient endocytosis, macro-autophagy and trafficking between endoplasmic reticulum and Golgi. Vesicles accumulating in these cells have a unique membrane phenotype associating the lysosomal marker LAMP1 with markers of the early secretory pathway, suggesting that they do not result from amplification of the pre-existing lysosomal compartment but rather arise from disease-related vesicular trafficking defects producing abnormal dead-end organelles related to but distinct from lysosomes. The generation and accumulation of abnormal vesicles unable to resolve could be a major cause of progressive intracellular storage in mucopolysaccharidosis IIIB neurons.