Oxytalan fibers are extracellular matrix components consisting of pure microfibrils. However, the mechanism whereby oxytalan fibers develop is not fully understood. We have previously reported that in human periodontal ligament (PDL) fibroblasts subjected to stretching stress, bundles of oxytalan fibers coalesce under the control of fibulin-5. Latent transforming growth factor-β binding protein 2 (LTBP-2) is known to bind to fibulin-5. The purpose of this study was to clarify the role of LTBP-2 in the coalescence of oxytalan fibers. We subjected PDL fibroblasts to stretching in order to examine the effects of LTBP-2 on the coalescence of oxytalan fibers in cell/matrix layers. Interaction of LTBP-2 with fibulin-5 was examined by immunoprecipitation assay, and changes in LTBP-2 deposition upon stretching were investigated by Western blotting and immunofluorescence assays. We used small interfering RNA against LTBP-2 in PDL cell culture and examined the appearance of oxytalan fibers on the basis of immunofluorescence. Stretching induced coalescence of oxytalan fibers, but did not affect LTBP-2 expression. The amount of extracellularly deposited LTBP-2 was decreased by about 70% as a result of stretching, compared with the control. LTBP-2 interacted with fibulin-5 on the fibers, and stretching decreased the amount of the LTBP-2 interacted with fibulin-5 by about 60%. Oxytalan fiber coalescence did not occur when LTBP-2 was suppressed by about 95%, whereas it occurred when LTBP-2 was suppressed by about 40%, fibulin-5 being colocalized with oxytalan fibers. These results suggest that LTBP-2, in response to tension stress, may negatively control the function of fibulin-5, thereby modulating the mechanism of oxytalan fiber coalescence. Copyright © Informa Healthcare USA, Inc.