Progesterone receptors (PRs) mediate proliferation during breast development and contribute to breast cancer progression, in part by synergizing with peptide growth factors. We have previously identified PR Ser294 as a key site for direct regulation of PR location, activity, and turnover in response to phosphorylation events. Herein, we sought to better understand how hormonal cross talk alters PR function. We demonstrate that progestins (R5020 and RU486) induce rapid (15 min) sumoylation of PR Lys388; sumoylation represses PR transcriptional activity on selected progesterone response element-driven and endogenous promoters and retards ligand-induced PR down-regulation. Consistent with this finding, we show that stabilized but weakly active phospho-mutant S294A PRs are heavily sumoylated. Conversely, desumoylated PR, created by mutation of PR Lys388 (K388R) or by overexpression of sentrin (SUMO)-specific protease desumoylating enzymes, are hypersensitive to low progestin concentrations. Combination of K388R and S294A mutations (KRSA double-mutant PR) rescues both transcription and turnover of impaired phospho-mutant (S294A) receptors. Notably, phosphorylation events antagonize PR-B but not PR-A sumoylation. Treatment of cells with epidermal growth factor or transient expression of activated mitogen-activated protein/ERK kinase kinase or cyclin-dependent protein kinase 2 induces PR-B Ser294 phosphorylation and blocks PR-B sumoylation, thereby derepressing receptor activity; PR-A is resistant to these events. Modulation of reversible PR sumoylation in response to diverse hormonal signals provides a mechanism for rapid isoform-specific changes in hormone responsiveness. In the context of elevated protein kinase activities, such as during mammary gland development or breast cancer progression, phosphorylated PR-B may be undersumoylated, transcriptionally hyperactive, and unstable/undetectable.