Drug resistance, including epirubicin-based therapeutic resistance, is one of the major reasons responsible for the unfavorable prognosis of patients diagnosed with breast cancer (BC). Acquired chemoresistance and metastatic properties have been identified to be closely associated with the process of epithelial-mesenchymal transition (EMT). Recently, dysregulation of long non-coding RNAs (lncRNAs) have been increasingly reported to perform promotive or suppressive functions in chemoresistance and EMT process in multiple cancers. However, relevant novel lncRNA participating in epirubicin resistance and EMT and its underlying molecular mechanisms remain unknown in BC. Herein, we established the epirubicin-resistant breast cancer cell subline (MCF-7/ADR), which presented mesenchymal phenotype and increased metastatic potential. A panel of differentially expressed lncRNAs, including 268 upregulated and 49 downregulated lncRNAs, were identified by high-flux microarray investigation in MCF-7 and MCF-7/ADR cells. The novel lncRNA NONHSAT101069 was significantly overexpressed in BC specimens, BC cell lines, and epirubicin-resistant cell sublines. The knockdown of NONHSAT101069 significantly repressed, whereas overexpression of NONHSAT101069 promoted the epirubicin resistance, migration, invasion and EMT process of BC cells both in vitro and in vivo. Further mechanism-related researches uncovered that NONHSAT101069 functioned as a ceRNA (competing endogenous RNA) via sponging miR-129-5p. Twist1 was a direct downstream protein of NONHSAT101069/miR-129-5p axis in BC cells. To conclude, NONHSAT101069 was upregulated in BC tissues and promoted epirubicin resistance, migration and invasion of BC cells via regulation of NONHSAT101069/miR-129-5p/Twist1 axis, highlighting its potential as an oncogene and a therapeutic biomarker for BC.