Paracrine factors, including growth factors and cytokines, released from cardiac myocytes following β-adrenergic receptor (β-AR) stimulation regulate cardiac fibroblasts. Activated cardiac fibroblasts have the ability to increase collagen synthesis, cell proliferation and myofibroblast differentiation, leading to cardiac fibrosis. However, it is unknown which β-AR subtypes and signaling pathways mediate the upregulation of paracrine factors in cardiac myocytes. In this study, we demonstrated that sustained stimulation of β-ARs significantly induced synthesis and secretion of growth factors, including connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), via the cAMP-dependent and protein kinase A (PKA)-dependent pathways. In addition, isoproterenol (ISO)-mediated synthesis and secretion of CTGF and VEGF through the β1-AR and β2-AR subtypes. Paracrine factors released by cardiac myocytes following sustained β-AR stimulation are necessary for the induction of cell proliferation and synthesis of collagen I, collagen III and α-smooth muscle actin (α-SMA) in cardiac fibroblasts, confirming that β-AR overstimulation of cardiac myocytes induces cardiac fibrosis by releasing several paracrine factors. These effects can be antagonized by β-blockers, including atenolol, metoprolol, and propranolol. Thus, the use of β-blockers may have beneficial effects on the treatment of myocardial fibrosis in patients with heart failure.