Long non-coding RNAs (lncRNAs) play key roles in human diseases, including cancer. Functional studies of the lncRNA HOTAIR (HOX transcript antisense RNA) provide compelling evidence for therapeutic targeting of HOTAIR in cancer, but targeting lncRNAs in vivo has proven to be difficult. In the current study, we describe a peptide nucleic acids (PNA)-based approach to block the ability of HOTAIR to interact with EZH2 and subsequently inhibit HOTAIR-EZH2 activity and resensitize resistant ovarian tumors to platinum. Treatment of HOTAIR-overexpressing ovarian and breast cancer cell lines with PNAs decreased invasion and increased chemotherapy sensitivity. Furthermore, the mechanism of action correlated with reduced nuclear factor-kappaB (NF-κB) activation and decreased expression of NF-κB target genes matrix metalloprotease 9 and interleukin 6. To deliver the anti-lncRNA to the acidic (pH approximately 6) tumor microenvironment, PNAs were conjugated to pH-low insertion peptide (pHLIP). Treatment of mice harboring platinum-resistant ovarian tumor xenografts with pHLIP-PNA constructs suppressed HOTAIR activity, reduced tumor formation and improved survival. This first report on pHLIP-PNA lncRNA targeting solid tumors in vivo suggests a novel cancer therapeutic approach.