Mutations in the VMA21 gene, encoding a V-ATPase chaperone protein, cause a X-linked myopathy with excessive autophagy and a congenital disorder of glycosylation altering muscle and liver, respectively. It remains unclear why mutations in VMA21 result in different diseases. To understand the clinical spectrum associated with VMA21 mutations, we investigated the consequences of the modulation of VMA21 in vitro and in vivo. First, we used lentiviral infection to generate stable cell lines of primary myoblasts, either 1) knock-out (KO) for Vma21, or 2) overexpressing (OE) the ubiquitous (VMA21-101) or muscle-specific (VMA21-120) isoforms of VMA21. Our results showed that overexpression of VMA21-120 perturbs the autophagic flux and the growth of primary myoblasts. In parallel, we failed to obtain Vma21KO and VMA21-101-OE cell lines, probably due to cell lethality. Second, we further characterized Vma21mKO mice recently developed in the lab to understand their premature lethality. We showed that skeletal muscle starts degenerating at 26 days of age in Vma21mKO mice, while autophagic lesions are already observed at 23 days. These results provide important information on the physiological role of VMA21 isoforms and XMEA pathogenesis.