Enzyme replacement therapy with laronidase is an established treatment for Mucopolysaccharidosis type I (MPS I), but its efficacy may be limited by the development of anti-drug antibodies, which inhibit cellular uptake of the enzyme. In a related disorder, infantile Pompe disease, immune tolerance induction with low-dose, short-course methotrexate appears to reduce antibody formation. We investigated a similar regimen using oral methotrexate in three MPS I patients. All patients developed anti-laronidase immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies, and they had clinically relevant levels of cellular uptake inhibition. We then explored several immune tolerance induction strategies in MPS I mice: (1) methotrexate, (2) combination of non-depleting anti-CD4 and anti-CD8 monoclonal antibodies, (3) methotrexate with anti-CD4 and anti-CD8 monoclonals, (4) anti-CD4 monoclonal, and (5) anti-CD8 monoclonal. Treated mice received 10 weekly laronidase injections, and laronidase was delivered with adjuvant on day 49 to further challenge the immune system. Most regimens were only partially effective at reducing antibody responses, but two courses of non-depleting anti-CD4 monoclonal antibody (mAb) ablated immune responses to laronidase in seven of eight MPS I mice (87.5%), even after adjuvant stimulation. Immune tolerance induction with methotrexate does not appear to be effective in MPS I patients, but use of non-depleting anti-CD4 monoclonal is a promising strategy.