Long non-coding RNAs (lncRNAs) play an important role in regulating several physiological processes and have been implicated in several pathologies including cancer. LncRNAs have been found to regulate key cellular pathways involved in cancer development, and their aberrant expression plays critical roles in the onset or progression of disease. The role of lncRNAs in breast cancer (BC) has become a hot topic of research in recent years. We previously showed that LINC00365 inhibits BC survival. In the current study, based on the important role of energy metabolism and HIF-1α for tumor cell proliferation, we investigated the role and mechanism of the LINC00365/HIF-1α axis in affecting tumor growth through glycolysis using the breast cancer cell lines MCF-7 and HCC-1937. We found that LINC00365 inhibited BC cell proliferation. Furthermore, LINC00365 overexpression suppressed aerobic glycolysis in BC cells. RNA-sequencing identified hypoxia-inducible factor-1α (HIF-1α), which has been linked with glycolysis and upregulates glycolysis-related genes, as a potential target gene of LINC00365. Accordingly, we found that LINC00365 overexpression resulted in decreased expression of key glycolytic enzymes such as downstream hexokinase 2 (HK2), recombinant pyruvate kinase isozymes M2 (PKM2) and lactate dehydrogenase A (LDHA). Our results suggest that targeting LINC00365 may reverse the glucose metabolism pattern of BC and effectively inhibit BC survival both in vitro and in vivo.