Human progesterone receptors (PR) rapidly activate cytosolic signaling pathways, in addition to their classical function as ligand-activated transcription factors. Using ER+/PR-B+ T47D breast cancer cells, we probed the role of progestin-stimulated rapid PR signaling in the transcriptional regulation of target genes involved in breast cancer cell proliferation. Epidermal growth factor receptor (EGFR) was rapidly activated after a 10-min treatment with R5020. Progestin induced EGFR-, c-Src-, and MAPK-dependent phosphorylation of PR-B on the MAPK consensus site, Ser345. Ser345-phosphorylated PR-B receptors strongly associated with specificity protein 1 (Sp1) transcription factors to regulate PR cell cycle (p21) and growth-promoting (EGFR) target genes whose promoters lack canonical progesterone response element sequences. Inhibitors of EGFR, c-Src, or MAPK activities blocked PR tethering to Sp1 and progestin-stimulated S-phase entry. Mutant PR-B receptors defective for c-Src binding (mPro) were not phosphorylated on Ser345 in response to progestin and failed to interact with Sp1. Hormoneinduced complexes containing Sp1 and wild-type PR-B, but not S345A or mPro PR-B, were recruited to Sp1 sites within the endogenous p21 promoter. Progestin-induced S-phase entry was attenuated in T47D cells containing wild-type PR-B and treated with EGFR, c-Src, or MAPK kinase inhibitors or in T47D cells stably expressing mPro or mutant DNA-binding domain PR-B. In sum, rapid progestin-activated PR signaling leads to PR Ser345 phosphorylation and tethering to Sp1. These events are critical for progestin-stimulated regulation of Sp1 target genes and breast cancer cell proliferation. Our data demonstrate the therapeutic potential for PR-targeted breast cancer treatment by exploiting multiple nodes along the PR signaling pathway, including PR-B, EGFR, c-Src, MAPK, or Sp1.