Misregulation of acid-base transport plays central roles in cancer development. We previously demonstrated the strong up-regulation of the Na(+),HCO3(-) cotransporter NBCn1 (SLC4A7) in MCF-7 breast cancer cells by a truncated, constitutively active ErbB2 (HER2) receptor, ΔNErbB2, and showed that NBCn1 expression and activity are increased in breast cancer tissue from patients. Here, we present the first in-depth characterization of an SLC4A7 promoter and identify its minimal ΔNErbB2-sensitive region. Inhibition or siRNA-mediated knockdown of PI3K, Akt1, ERK1/2, or Src decreased the NBCn1 protein level in ΔNErbB2-expressing MCF-7 cells by ∼50, 60, 30 and 35%, respectively. Further, knockdown of the transcription factor Krüppel-like factor 4 (KLF4) reduced NBCn1 protein expression by ∼40%, and KLF4 overexpression increased NBCn1 expression by 50-80%. In contrast, knockdown of the closely related transcription factor specificity protein 1 (Sp1) or transfection with dominant-negative Sp1 increased NBCn1 expression by ∼35 and ∼50%, respectively. NBCn1 expression was also increased by stimulation of full-length ErbB1, -2, and -3 receptors in SKBr3 cells (1.5- and 2-fold by NRG1 or EGF, respectively) or after their exogenous expression in MCF-7 cells. Finally, stimulation with NRG1 or EGF more than doubled acid extrusion capacity in SKBr3 cells. In conclusion, NBCn1 is strongly upregulated by ErbB receptor signaling in a manner involving opposite effects of KLF4 and Sp1, transcription factors with central roles in cancer development. ErbB-induced up-regulation of NBCn1-mediated acid extrusion may play important physiological and pathophysiological roles in the breast epithelium and other tissues with high ErbB receptor levels.-Gorbatenko, A., Olesen, C. W., Mørup, N., Thiel, G., Kallunki, T., Valen, E., Pedersen, S. F. ErbB2 upregulates the Na(+),HCO3(-)-cotransporter NBCn1/SLC4A7 in human breast cancer cells via Akt, ERK, Src, and Krüppel-like factor 4.