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Fibrosis is characterized by hardening, overgrowth, and development of scars in various tissues as a result of faulty reparative processes, diseases, or chronic inflammation. During the fibrotic process in the corneal stroma of the eye, the resident cells called keratocytes differentiate into myofibroblasts, specialized contractile fibroblastic cells that produce excessive amounts of disorganized extracellular matrix (ECM) and pro-fibrotic components such as alpha-smooth muscle actin (alpha-SMA) and fibronectin. This study aimed to elucidate the role of substance P (SP), a neuropeptide that has been shown to be involved in corneal wound healing, in ECM production and fibrotic markers expression in quiescent human keratocytes, and during the onset of fibrosis in corneal fibroblasts, in an in vitro human corneal fibrosis model. We report that SP induces keratocyte contraction and upregulates gene expression of collagens I, III, and V, and fibrotic markers: alpha-SMA and fibronectin, in keratocytes. Using our in vitro human corneal fibrosis model, we show that SP enhances gene expression and secretion of collagens I, III, and V, and lumican. Moreover, SP upregulates gene expression and secretion of alpha-SMA and fibronectin, and increases contractility of corneal fibroblasts during the onset of fibrosis. Activation of the preferred SP receptor, the neurokinin-1 receptor (NK-1R), is necessary for the SP-induced pro-fibrotic changes. In addition, SP induces the pro-fibrotic changes through activation of the RhoA/ROCK pathway. Taken together, we show that SP has a pro-fibrotic effect in both quiescent human keratocytes and during the onset of fibrosis in an in vitro human corneal fibrosis model.