Acidosis is an important factor on tumor development, but little is known about activated mechanisms of action. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which triggers non-genomic effects through c-Src. Considering that AhR/c-Src axis promotes breast cancer progression when is activated by ligand, this makes it a possible target to be induced by the acidic tumor microenvironment. Our aim was to study the effect of extracellular pH (pHe) 6.5 on AhR/c-Src axis and its correlation with cell migration and metalloproteases (MMP)-2 and 9 activities, using two breast cancer cell lines (MDA-MB-231 and LM3) and the mammary epithelial cells NMuMG. We found that acidosis induces c-Src phosphorylation only in breast cancer cells through AhR, since it was prevented by the AhR inhibitor 4,7-o-phenanthroline (PHE). In addition, the pHe 6.5 was blocked with PHE or the c-Src inhibitor PP2. Cytosolic pH (pHi) was measured in MDA-MB-231 cells treated with pHe 6.5, founding a reduction from 7.6 to 6.9. Amiloride is an inhibitor of the Na+/H+ exchange 1 protein that is known to reduce pHi. MDA-MB-231 treatment with amiloride enhances c-Src phosphorylation in an AhR-dependent manner, suggesting that the reduction in pHi could be involved in AhR/c-Src activation. Evidence suggests that acidosis induces a pro-migratory phenotype in breast cancer cells through AhR/c-Src signaling.