Purpose To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-β-related cytokine in human trabecular meshwork (TM) cells in the context of aqueous humor (AH) outflow and IOP. Methods Regulation of expression by external cues, and the distribution and secretion of GDF-15 by human TM primary cell cultures, and the effects of recombinant (r) GDF-15 on TM cell contractile characteristics, actin cytoskeleton, cell adhesion, extracellular matrix (ECM), α-smooth muscle actin (αSMA), SMAD signaling, and gene expression were determined by immunoblot, immunofluorescence, mass spectrometry, cDNA microarray, and real-time quantitative PCR (RT-qPCR) analyses. Results Growth differentiation factor-15, a common constituent of ECM derived from the human TM cells, was confirmed to be distributed throughout the conventional aqueous humor outflow pathway of the human eye. Growth differentiation factor-15 protein levels were significantly increased in human TM cells in response to TGF-β2, dexamethasone, endothelin-1, lysophosphatidic acid, TNF-α, IL-1β treatment, and by cyclic mechanical stretch. Stimulation of human TM cells with rGDF-15 caused a significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, SMAD signaling, gene expression, and the levels of αSMA and ECM proteins. Conclusions The results of this study, including a robust induction of GDF-15 expression by several external factors known to elevate IOP, and rGDF-15-induced increase in contractility, cell adhesion, and the levels of ECM proteins and αSMA in TM cells, collectively suggest a potential role for GDF-15 in homeostasis and dysregulation of AH outflow and IOP in normal and glaucomatous eyes, respectively.