The human JC polyomavirus (JCPyV) is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). The Mad-1 prototype strain of JCPyV uses the glycan lactoseries tetrasaccharide c (LSTc) and serotonin receptor 5-HT2A to attach to and enter into host cells, respectively. Specific residues in the viral capsid protein VP1 are responsible for direct interactions with the α2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are hypothesized to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as pentamers in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structure determination revealed that these pentamers are well folded but no longer bind to LSTc due to steric clashes in the sialic acid-binding site. Reconstitution of the mutations into JCPyV pseudoviruses allowed us to directly quantify the infectivity of the mutants in several cell lines. The JCPyV pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs and render these viruses noninfectious. IMPORTANCE Infection with human JC polyomavirus (JCPyV) is common and asymptomatic in healthy individuals, but during immunosuppression, JCPyV can spread from the kidney to the central nervous system (CNS) and cause a fatal, demyelinating disease, progressive multifocal leukoencephalopathy (PML). Individuals infected with HIV, those who have AIDS, or those receiving immunomodulatory therapies for autoimmune diseases are at serious risk for PML. Recent reports have demonstrated that viral isolates from PML patients often have distinct changes within the major capsid protein. Our structural-functional approach highlights that these mutations result in abolished engagement of the carbohydrate receptor motif LSTc that is necessary for infection. Viruses with PML-associated mutations are not infectious in glial cells, suggesting that they may play an alternative role in PML pathogenesis.