Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear β-actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear β-actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear β-actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular β-actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and β-actin in developing mammary end buds. These data indicate a novel role for nuclear β-actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.