The lipid metabolic enzymes are considered candidate therapeutic targets for breast cancer. Long-chain acyl-coenzyme A (CoA) synthase (ACSL) is one of lipid metabolic enzymes and converts free-fatty acid to fatty acid-CoA. Five ACSL isoforms including ACSL1, ACSL3, ACSL4, ACSL5 and ACSL6 are identified in human. High ACSL4 expression has been observed in aggressive breast cancer phenotype. However, the role of other isoforms is still little-known. We therefore, analyzed the expression of ACSL isoforms in each subtype of breast cancer within METABRIC dataset and cancer cell line encyclopedia dataset. The expression levels of ACSL1, ACSL4 and ACSL5 in estrogen receptor (ER)-negative group were higher than that in ER-positive group. Similar expression pattern was detected among breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-231 (ER-negative). Treatment of ACSL inhibitor triacsin C which inhibited enzyme activity of ACSL 1, 3, 4 and 5 suppressed cell growth of MCF-7 and MDA-MB-231. Our results further showed that high ACSL5 expression was associated with good prognosis in patients with both ER-positive and ER-negative breast cancer through KM plotter analysis. These results suggest that ACSL1, ACSL4 and ACSL5 expression is regulated by ER signaling pathways and ACSL5 is a potential novel biomarker for predicting prognosis of breast cancer patients.