Trabecular meshwork (TM) cells have widely been used as an in vitro model for glaucoma research. However, primary TM cells suffer the disadvantages of limited cell numbers and slow rates of proliferation. We discovered a spontaneously transformed bovine TM (BTM) cell line, BTM-28T. This cell line proliferated rapidly in low-glucose culture medium but also demonstrated contact inhibition in high-glucose culture medium. BTM-28T cells expressed key TM cell markers including α-smooth muscle actin (α-SMA), laminin and collagen IV (col IV). Also, 100 nM dexamethasone (DEX) enhanced the formation of cross-linked actin networks (CLANs) in confluent BTM-28T cell cultures. Transforming growth factor beta 2 (TGFβ2) induced the expression of fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF) in our cell cultures. This cell line will be helpful to better understand the aqueous humor outflow pathway as related to the pathophysiology of glaucoma.