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The intervertebral disk (IVD) is a composite structure essential for spine stabilization, load bearing, and movement. Biomechanical factors are important contributors to the IVD microenvironment regulating joint homeostasis; however, the cell type-specific effectors of mechanotransduction in the IVD are not fully understood. The current study aimed to determine the effects of cyclic tensile strain (CTS) on annulus fibrosus (AF) cells and identify mechano-sensitive pathways. Using a cell-type specific reporter mouse to differentiation NP and AF cells from the murine IVD, we characterized AF cells in dynamic culture exposed to CTS (6% strain) at specific frequencies (0.1 Hz, 1.0 Hz, or 2.0 Hz). We demonstrate that our culture model maintains the phenotype of primary AF cells and that the bioreactor system delivers uniform biaxial strain across the cell culture surface. We show that exposure of AF cells to CTS induces cytoskeleton reorganization resulting in stress fiber formation, with acute exposure to CTS at 2.0 Hz inducing a significant yet transient increase ERK1/2 pathway activation. Using SYBPR-based qPCR to assess the expression of extracellular matrix (ECM) genes, ECM-remodeling genes, candidate mechano-sensitive genes, inflammatory cytokines and cell surface receptors, we demonstrated that exposure of AF cells to CTS at 0.1 Hz increased Acan, Prg4, Col1a1 and Mmp3 expression. AF cells exposed to CTS at 1.0 Hz showed a significant increase in the expression of Acan, Myc, and Tnfα. Exposure of AF cells to CTS at 2.0 Hz induced a significant increase in Acan, Prg4, Cox2, Myc, Fos, and Tnfα expression. Among the cell surface receptors assessed, AF cells exposed to CTS at 2.0 Hz showed a significant increase in Itgβ1, Itgα5, and Trpv4 expression. Our findings demonstrate that the response of AF cells to CTS is frequency dependent and suggest that mechanical loading may directly contribute to matrix remodeling and the onset of local tissue inflammation in the murine IVD.