Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post-translationally modified by a series of specific enzymes. Among the collagen-modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross-linking and lysyl hydroxylases (LHs) to regulate the cross-linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen-modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra-peritoneal injection of a LOX inhibitor, β-aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL-derived cells. The mechanical loading also elevated formation of collagen α-chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2-positive cells was observed only at the bone-side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up-regulating collagen-modifying enzymes and subsequent collagen cross-linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926-933, 2016. © 2015 Wiley Periodicals, Inc.