Cysteine as well as glutamine and alanine are critical substrates for synthesis of glutathione (GSH).Glutamine and alanine, but not cysteine, are most upregulated intermediate metabolites in cancer cells, suggesting cysteine uptake is increased. However, it's not clear how the uptake of cysteine is regulated. Here, we report that the cysteine carrier SLC3A1 is upregulated in breast cancer cells, and its expression levels are correlated with clinical stages and patients'survival. In breast cancer cells, overexpression of SLC3A1 accelerates the uptake of cysteine, which in turn increases the concentration of reductive GSH and the GSH/GSSH ratio and concomitantly decreases the cellular level of ROS (reactive oxygen species).Consequently, overexpression of SLC3A1 promotes the tumorigenesis of breast cancer cells, whereas knocking-down or inhibition of SLC3A1 decreases tumorigenesis of breast cancer cells in mice. Moreover, SLC3A1 inhibitor Sulfasazine suppressed tumor growth and abolished dietary NAC-promoted tumor growth. Mechanistically, our data manifest that ROS catalyzes thiol into sulfenic acid at the HC (X) 4EXV motif of serine/threonine phosphatase PP2Ac which is similar as the ROS-induced sulfenylation at the HC (X) 5RS/T motif of protein tyrosine phosphatases (PTPs); however, unlike PTPs, sulfenylation increases the stability and activity of PP2Ac rather than deactivating PTPs. SLC3A1 activates the Akt pathway through decreasing the sulfenylation and stability of protein phosphatase PP2Ac. Collectively, our data demonstrate that SLC3A1-mediated increase of cysteine uptake promotes the tumorigenesis of breast cancer cells, and SLC3A1 determines breast cancer cell response to antioxidant N-acetylcysteine, suggesting SLC3A1 is a potential therapeutic target for breast cancer.