Approximately 28,000 long noncoding RNAs (lncRNAs) are transcribed from the human genome, with some localized in the nucleus. These lncRNAs have been revealed to play vital and diverse roles in nuclear functions, including transcriptional regulation, DNA damage repair, and maintenance of nuclear compartments. Importantly, dysregulation of these lncRNAs has been linked to many human diseases, especially cancer. Here we show that TUG1, a highly expressed lncRNA in many types of cancers, plays a crucial role in resolving pathogenic R-loops, a source of DNA damage and genome instability. Depleting TUG1 by antisense oligonucleotides (ASO) resulted in an overabundant R-loops and DNA damage, leading to significant inhibition of cell proliferation. In a glioblastoma xenograft mouse model, treatment with TUG1 ASO, coupled with a tumor-specific drug delivery system, substantially inhibited tumor growth. Our study unveils a novel role of lncRNA in maintaining genome stability in cancer cells and provides a strong rationale for targeting TUG1 as a potent therapeutic approach for cancer treatment.