Focal segmental glomerulosclerosis (FSGS) is characterized by typical sclerosis but also shows other non-sclerotic lesions that provide prognostic informations. The glomerular epithelial hyperplasia lesion (EPHL) that develops earlier than the sclerotic lesions is a key determinant of progression of FSGS. However, the relationship among EPHL, glomeular sclerosis, and macrophage infiltration in FSGS is unclear, and the EPHL-associated markers helpful for prognosis of FSGS have still not been completely identified. Here, we performed clinicopathologic, immunochemical, and molecular analyses to examine whether osteopontin (OPN), a macrophage chemokine, is an injury marker of EPHLs correlating with glomerular sclerosis and macrophage mobilization. First, the FSGS model was induced in Balb/c mice by a single injection of adriamycin, and consecutive sclerosis changes were evaluated. In parallel, we used reverse transcription-polymerase chain reaction and Western blot analyses to determine levels of OPN in isolated glomeruli and urine, respectively. Immunohistochemistry was applied to assess the OPN expression in EPHLs and macrophage infiltration around the glomeruli. Our results showed that, within glomeruli, OPN expressed restrictedly within EPHL; the OPN mRNA and protein of glomeruli increased on day 11, correlating well with the early EPHL, and following sclerosis and macrophage infiltration. In addition, immunohistochemistry (IHC) staining of OPN greatly highlighted early glomerular EPHLs, helping microscopic identification of EPHLs. We propose that the OPN expression in EPHLs could contribute to the progression of FSGS by recruiting macrophage toward the compromised glomeruli. Detection of OPN in glomeruli and urine could be helpful in prognosis of FSGS.