Estrogen Receptor β (ERβ) is a member of the nuclear receptor family of homeostatic regulators that is frequently lost in breast cancer (BC), where its presence correlates with a better prognosis and a less aggressive clinical outcome of the disease. Contrary to ERα, its closest homolog, ERβ shows also significant estrogen-independent activities, including the ability to inhibit cell cycle progression and to regulate gene transcription in the absence of the ligand. Investigating the nature and extent of this constitutive activity of ERβ in BC MCF-7 and ZR-75.1 cells by miRNA-Seq, we identified 30 miRNAs differentially expressed in ERβ+ vs ERβ- cells in the absence of ligand, including upregulated oncosuppressor miRs, such miR-30a. In addition, a significant fraction of >1,600 unique proteins identified in MCF-7 cells by iTRAQ (isobaric Tag for Relative and Absolute Quantitation) quantitative proteomics was either increased or decreased by ERβ, revealing regulation of multiple cell pathways by ligand-free receptor. Transcriptome analysis show that for a large number of proteins regulated by ERβ the corresponding mRNAs are unaffected, including a large number of putative targets of ERβ-regulated miRNAs, indicating a central role of miRNAs in mediating BC cell proteome regulation by ERβ. Expression of a mimic of miR-30a-5p, a direct target and downstream effector of ERβ in BC, led to the identification of several target transcripts of this miRNA, including 11 encoding proteins whose intracellular concentration is significantly affected by unliganded receptor. These results demonstrate a significant effect of ligand-free ERβ on BC cell functions via modulation of the cell proteome and suggest that miRNA regulation may represent a key event in the control of the biological and clinical phenotype of hormone-responsive BC by this nuclear receptor.