This study aimed at exploring the role of α-enolase (ENO1) in proliferation, invasion, and cell apoptosis in MDA-MB-231 and MCF-7 breast cancer human cells, to provide a theoretical basis for the clinical treatment of breast cancer. MDA-MB-231 and MCF-7 cells were randomly divided into five groups: normal control group (Control group), negative control group (negative control virus, NC group), and shENO1 (sh1, sh2, and sh3) group, respectively. The expressions of ENO1 mRNA and protein were measured by Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Cell proliferation, cell invasion ability, and cell apoptosis rate were detected by methyl thiazolyl tetrazolium (MTT) assay, transwell invasion assay, and flow cytometer, respectively. The expressions of the proteins correlated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were analyzed by Western blot. In MDA-MB-231 and MCF-7 cells, the gene and protein expressions of ENO1 in the three sh groups in MDA-MB-231 and MCF-7 cells were significantly lower than those in control group and NC group. In MDA-MB-231 and MCF-7 cells, the gene and protein expressions of ENO1 in the three sh groups were significantly lower than those in control group and NC group. Compared with NC group, the proliferation activity, invasion ability, and apoptosis rate of shENO1 group were significantly decreased (p < 0.01). PI3K and Akt protein levels in shENO1 group were significantly downregulated (p < 0.01). Bcl-2 protein expression was markedly upregulated (p < 0.01), meanwhile Bax protein revealed a significant reduction (p < 0.01). The results revealed that silencing ENO1 reduced proliferation activity, invasion ability, and apoptosis rate of breast cancer cells by decreasing the phosphorylation of PI3K and Akt pathway. Our results suggested that ENO1 may be a potential therapeutic target in breast cancer.