Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of the resistance are still unclear. JAK 1/2 inhibitors (such as ruxolitinib and JAK Inhibitor 1) strongly stimulate VSV replication and oncolysis in all resistant cell lines, but only partially improve susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication, rather than on receptor specificity, with several ubiquitously expressed cell-surface molecules to play a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDACs. Our data show a dramatically weaker attachment of VSV to HPAF-II, the most resistant human PDAC cell line. Although sequence analysis of LDLR mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels, but did not improve VSV attachment or LDL uptake in HPAF-II. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and polybrene or DEAE-dextran successfully broke the resistance of HPAF-II to VSV by simultaneously improving VSV attachment and replication.IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC). Although VSV is effective against most PDACs, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDACs. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (inhibits antiviral signaling) successfully broke the resistance of HPAF-II to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple combination approach could be used in the future to treat PDAC tumors highly resistant to OV therapy.