Pompe disease is a rare glycogen storage disorder caused by a deficiency in the lysosomal enzyme acid α-glucosidase, which leads to muscle weakness, cardiac and respiratory failure, and early mortality. Alglucosidase alfa, a recombinant human acid α-glucosidase, was the first approved treatment for Pompe disease, but its uptake into skeletal muscle via the cation-independent mannose-6-phosphate (M6P) receptor (CIMPR) is limited. Avalglucosidase alfa has received marketing authorization in several countries for infantile-onset and/or late-onset Pompe disease. This recently approved enzyme replacement therapy (ERT) was glycoengineered to maximize CIMPR binding through high-affinity interactions with ~7 bis-M6P moieties. Recently, small molecules like the glucosylceramide synthase inhibitor miglustat were reported to increase the stability of recombinant human acid α-glucosidase and it was suggested that an increased serum half-life would result in better glycogen clearance. Here, the effects of miglustat on alglucosidase alfa and avalglucosidase alfa stability, activity, and efficacy in Pompe mice were evaluated. While miglustat increased the stability of both enzymes in fluorescent protein thermal shift assays and when incubated in neutral pH buffer over time, it reduced their enzymatic activity by ~50%. Improvement in tissue glycogen clearance and transcriptional dysregulation in Pompe mice correlated with M6P levels, but not with miglustat co-administration. These results further substantiate the crucial role of CIMPR binding in lysosomal targeting of ERTs. Significance Statement This work describes important new insights into the treatment of Pompe disease using currently approved ERTs co-administered with miglustat. Though miglustat increased the stability of ERTs in vitro, there was no positive impact to glycogen clearance and transcriptional correction in Pompe mice. However, increasing M6P levels resulted in increased cell uptake in vitro, and increased glycogen clearance and transcriptional correction in Pompe mice, further underscoring the crucial role of CIMPR-mediated lysosomal targeting for ERTs.