Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase, results in accumulation of glycosaminoglycans (GAGs) including heparan sulfate (HS). In patients with severe form MPS II, current intravenous enzyme replacement therapy (ERT) is not effective in improving brain pathology. This study aimed to examine the efficacy of intracerebroventricular (ICV) ERT of idursulfase-beta (IDS-β). We performed single ICV injections of three different doses (3, 10, and 30 μg) of IDS-β in IDS KO mice. The contents of tissue GAGs, HS in CSF and brain was measured at every 7 days after a single injection. After demonstrating the efficacy and dose-responsiveness of single injection, the long-term efficacy was elucidated with 30 μg of IDS-β ICV injections every 4 weeks for 6 times. The long-term efficacy was assessed by HS contents in CSF and brain, brain MRI, histologic exam, and behavioral tests. We also evaluated the correlation between CSF HS and brain HS or GAGs. In single injection study, the GAGs contents in the brain and somatic tissues of 30-μg IDS-β ICV ERT group were significantly reduced and the reduction was maintained for 28 days. The HS content was also significantly reduced in both the CSF and brain tissue of all IDS-β-treated groups. Furthermore, HS concentration in the CSF was significantly correlated with brain HS and brain GAGs. In long-term injection study, the HS concentration of brain and CSF was significantly reduced and correlated. The mice with ICV ERT had improvements of ventriculomegaly in the brain MRI and showed reduction of lysosomal storages in histologic exam. The significant improvements in memory/learning function were observed in open field and fear conditioning tests. ICV ERT with 30-μg of IDS-β produced significant improvements of biochemical, histological and functional parameters in MPS II mice, suggesting a feasible clinical therapeutic option. We also suggest that CSF HS concentration could be a useful biomarker for brain storage in MPS II patients with CNS involvement.