The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAR) exhibits basolateral localization， the eight exon-encoded isoform (CAR) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms， CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAR cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized， and doxycycline-induction of CAR restored sensitivity to adenovirus. These data reinforce the importance of CAR in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.