Abstract Renal fibrosis arises by the generation of matrix-producing fibroblasts and myofibroblasts through the epithelial–mesenchymal transition (EMT), a process in which epithelial cells undergo a transition into a fibroblast phenotype. A key feature of the EMT is the reorganization of the cytoskeletons, which may involve the Ca2+-binding protein S100A16, a newly reported member of the S100 protein family. However, very few studies have examined the role of S100A16 in renal tubulointerstitial fibrosis. In this study, S100A16 expression was examined by immunohistochemical staining of kidney biopsy specimens from patients with various nephropathies and kidney tissues from a unilateral ureteral obstruction (UUO) mouse model. Renal histological changes were investigated in S100A16Tg, S100A16+/−, and WT mouse kidneys after UUO. The expression of epithelia marker E-cadherin, mesenchymal markers N-cadherin, and vimentin, extracellular matrix protein, and S100A16, as well as the organization of F-actin, were investigated in S100A16 overexpression or knockdown HK-2 cells. Mass spectrometry was employed to screen for S100A16 binding proteins in HK-2 cells. The results indicated that S100A16 is high expressed and associated with renal tubulointerstitial fibrosis in patient kidney biopsies and in those from UUO mice. S100A16 promotes renal interstitial fibrosis in UUO mice. S100A16 expression responded to increasing Ca2+ and interacted with myosin-9 during kidney injury or TGF-β stimulation to promote cytoskeleton reorganization and EMT progression in renal tubulointerstitial fibrosis. Therefore, S100A16 is a critical regulator of renal tubulointerstitial fibroblast activation and is therefore a potential therapeutic target for the treatment of renal fibrosis.