Glioblastoma (GBM) is an aggressive and therapy resistant central nervous system tumor. Wnts are involved in a plethora of cellular functions such as cell proliferation, differentiation, and migration. Deregulation of Wnt/β-catenin signaling is frequently observed in GBM, leading to glioma stem cell (GSC) maintenance, invasion, and resistance to therapy. Moreover, the cellular prion protein (PrPC) may interact with β-catenin and activate transcriptional activity of the β-catenin/TCF complex. PrPC plays essential roles in proliferation and differentiation, and was shown to upregulate the activity of Wnt/β-catenin signaling on intestinal progenitors. Our group has demonstrated that PrPC is overexpressed in GBM and that this leads to a higher proliferation rate and stemness maintenance. We have also demonstrated that silencing PrPC decreases the expression of stem cell markers, self-renewal, and tumorigenesis of GSCs. To further explore this connection between Wnt signaling and PrPC, we generated PrPC KO human U87 and U251 GBM cells with the CRISPR-Cas9 system. We performed RNA sequencing and subsequently validated differentially expressed genes (DEGs) by RT-qPCR. DESeq2 analysis comparing PrPC-KO GBM cells with wild-type controls resulted in 1,295 DEGs for U87 cells and 363 DEGs for U251 cells. Of those U87 and U251 DEGs, 28 and 10, respectively, are part of signaling pathways regulated by Wnt proteins. We chose to validate the upregulated DEGs CAMK2A, NFATC4, WISP1, PPP2R2B, SFRP2; and the downregulated DEGs RAC3 and SHH. In U87 GBM cells, expression patterns of CAMK2A, NFATC4, WISP1, PPP2R2B and SHH matched what we found on the RNAseq analysis, while RAC3 expression did not differ. In addition, SFRP2 was downregulated in U87 cells but appeared as an upregulated DEG. In U251 cells, expression of NFATC4 and CAMK2A was downregulated, and expression of RAC3 was upregulated. Interestingly, NFATC4, CAMK2A and RAC3 were not U251 DEGs in our RNAseq analysis, but their expression is altered in the PrPC-KO group. We will analyze the expression of SFRP2, PPP2R2B, WISP1 and SHH on U251 GBM cells. Our results indicate that the Wnt-Ca2+ pathway may be activated in U87 PrPC-KO cells, due to the increased expression of NFATC4 and CAMK2A. Regarding the canonical Wnt signaling, it appears to be a balance in the expression of genes that activate or inhibit these pathways in PrPC-KO cells, since WISP1 and PPP2R2B were upregulated and SFRP2 was downregulated. Importantly, expression of SHH was downregulated in U87 PrPC-KO cells. Aberrant activation of the Sonic Hedgehog signaling is implicated in the invasive capacity of GBM. We will also evaluate the status of Wnt signaling on PrPC-KO cells through Western blotting. Nevertheless, our results demonstrate that the Wnt signaling is affected by the KO of PrPC in GBM and further corroborate the importance of PrPC in Wnt signaling for GBM biology. Supported by FAPESP (2019/14952-0) and Capes.