Background: To explore the role of Nei endonuclease VIII-like 3 (NEIL3) in breast cancer (BC) cell proliferation and motility and its underlying mechanism. Methods: BC cell lines were analyzed. NEIL3 expression levels in BC tissues was assessed using the UALCAN and Kaplan-Meier Plotter databases. NEIL3 knockdown was performed using siRNAs, and its effects on cell growth, motility, Epithelial-Mesenchymal Transition (EMT) markers, and the Phosphoinositide 3-Kinase/Protein Kinase B/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) axis were evaluated using Immunoblotting, cell counting kit-8 (CCK-8) assays, colony formation, Transwell as well as wound healing assays. Results: NEIL3 was highly expressed in BC tissues and cell lines. NEIL3 knockdown inhibited cell growth, motility and reduced EMT marker expression. It also decreased phosphorylation levels of proteins, indicating inhibition of the PI3K/Akt/mTOR axis. Conclusions: NEIL3 mediates BC cell proliferation as well as motility by activating the PI3K/Akt/mTOR axis. Targeting NEIL3 may offer a new approach to combat BC.