Background: Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program.Results: Tetracycline-regulated expression of CTGF/CCN2 in HC11 cells enhanced multiple markers of lactogenic differentiation including β-casein transcription and mammosphere formation. In a separate measure of mammary differentiation the addition of CTGF/CCN2 to cultures of MCF10A cells increased the development of acini in vitro. In HC11 cells the elevated levels of CTGF/CCN2 diminished the requirement for extracellular matrix proteins in the activation of β-casein transcription, indicating that CTGF/CCN2 contributed to lactogenic differentiation through the regulation of matrix dependent cell adhesion. CTGF/CCN2 expression in HC11 cells increased expression of extracellular matrix proteins and integrins, enhanced the formation of focal adhesion complexes, and increased survival signaling. In addition, HC11 cells adhered to immobilized CTGF/CCN2 and this was inhibited by function-blocking antibodies to the integrins α6 and β1, and to a lesser degree by antibody to β3 integrin.Conclusions: CTGF/CCN2 expression in HC11 cells led to an increase in multiple markers of lactogenic differentiation. The mechanisms by which CTGF/CCN2 contributed to lactogenic differentiation include direct binding of CTGF/CCN2 to integrin complexes and CTGF/CCN2-induced matrix protein expression resulting in elevated integrin functionality.