Prolactin (PRL) and its receptor the PRLR are implicated in breast cancer invasiveness, although their exact roles remain controversial. The Na(+)/H(+) exchanger NHE1 plays essential roles in cancer cell motility and invasiveness, but the PRLR and NHE1 have not previously been linked. Here, we show that in T47D human breast cancer cells, which express high levels of PRLR and NHE1, exposure to PRL led to activation of Janus Kinase-2 (JAK2)/Signal transducer and activator of transcription- 5 (STAT5)-, Akt, and Extracellular Signal Regulated Kinase-1/2 (ERK1/2) signaling, and rapid formation of peripheral membrane ruffles, known to be associated with cell motility. NHE1 was present in small ruffles prior to PRL treatment and was further recruited to the larger, more dynamic ruffles induced by PRL exposure. In PRL-induced ruffles, NHE1 colocalized with activated Akt, ERK1/2, and the ERK effector p90Ribosomal S Kinase (p90RSK), known regulators of NHE1 activity. Stimulation of T47D cells with PRL augmented p90RSK activation, Ser703-phosphorylation of NHE1, NHE1-dependent intracellular pH recovery, pericellular acidification, and NHE1-dependent invasiveness. NHE1 activity and localization to ruffles were attenuated by inhibition of Akt and/or ERK1/2. In contrast, non-cancerous MCF10A breast epithelial cells expressed NHE1 and PRLR at lower levels than T47D cells and their stimulation with PRL induced neither NHE1 activation nor NHE1-dependent invasiveness. In conclusion, we show for the first time that PRLR activation stimulates breast cancer cell invasiveness via activation of NHE1. We propose that PRL-induced NHE1 activation and resulting NHE1-dependent invasiveness may contribute to the metastatic behavior of human breast cancer cells.