Cellular prion protein (PrP<sup>C</sup>) is a glycoprotein tethered to the plasma membrane via a GPI-anchor, and it plays a crucial role in prion diseases by undergoing conformational change to PrP<sup>Sc</sup>. To generate a knock-in (KI) mouse model expressing bank vole PrP<sup>C</sup> (BVPrP<sup>C</sup>), a KI targeting construct was designed. However, a Prnp gene sequence that encodes PrP<sup>C</sup> lacking seven C-terminal amino acid residues of the GPI-anchoring signal sequence (GPI-SS) was unintentionally introduced into the construct. The resulting KIBVPrP248 mice exhibited very low PrP<sup>C</sup> expression and resistance to prion infection. To investigate the underlying mechanism of reduced PrP<sup>C</sup> expression, RK13 cells expressing either full-length GPI-SS (BVPrP255) or truncated GPI-SS (BVPrP248) and KIBVPrP248 mice were analyzed. In RK13-BVPrP248 cells, PrP<sup>C</sup> protein levels were nearly ten-fold lower than in RK13-BVPrP255 cells, mimicking the extremely low PrP<sup>C</sup> expression of the KIBVPrP248 mice. The abundance, stability, and translational efficiency of the Prnp mRNA were not the primary causes for the low PrP<sup>C</sup> expression in RK13-BVPrP248 cells. A pharmacological analysis revealed that BVPrP248 underwent enhanced degradation via the ER-associated degradation pathway, with increased PrP ubiquitination detected in both the cell and animal models. An immunofluorescence analysis showed that BVPrP248 was mislocalized to the ER, co-localizing with Grp78, an ER chaperone. Although mislocalization of BVPrP248 under the transient overexpression condition led to mild activation of the unfolded protein response in RK13-BVPrP248 cells, low-level chronic expression of BVPrP248 in stable transfectants and KIBVPrP248 mice did not facilitate such events. These findings suggested that the C-terminal GPI-SS of PrP<sup>C</sup> plays a critical role in PrP<sup>C</sup> biogenesis.