A key event in prion diseases is the conformational conversion of a cellular normal form of the prion protein (PrP^C) to disease-associated conformers (PrP^Sc). Polymorphisms or mutations in the human prion protein gene (PRNP) are associated with acquired or hereditary human prion diseases. When human prion protein (PrP) variants are characterized by cDNA expression systems in cultured cells, the endogenous prion protein may affect the behavior of ectopically expressed prion variants. To eliminate this issue, we herein created a PRNP-disrupted mutant cell line from human neuroblastoma BE(2)-M17 cells. No morphological differences were observed between PRNP-null and parental cells. We stably introduced cDNAs encoding E196K-PrP and E200K-PrP (both of which are Creutzfeldt-Jakob disease (CJD)-related mutants), a non-pathogenic E219K-PrP, and wild-type control PrP into PRNP-null mutant cells using the PiggyBac system, and found that the glycosylation pattern of E196K-PrP differed from that of other PrPs. We also found that non-glycosylated PrPs for E196K- and E200K-PrPs had distinct electrophoretic mobilities from that of WT-PrP, and E219K-PrPs exhibited slightly different mobilities in polyacrylamide gel electrophoresis under endogenous PrP-null conditions, demonstrating that the PRNP-disrupted human neuroblastoma cell line serves as a useful tool for examining PrPs with mutations or polymorphisms.