Mitotically inactivated feeder cells such as mouse embryonic fibroblast (MEFs) cells have been widely applied for physical and physiological support in the pluripotency maintenance of human pluripotent stem cells (hPSCs). However, accurate supporting mechanism or factors of feeder cells are poorly understood. Here, we isolated differentially expressed genes between wild-type MEFs and mitotically inactivated MEFs (miMEFs) by employing annealing control primer-based GeneFishing polymerase chain reaction. We identified a secreted protein acidic cysteine-rich glycoprotein (SPARC) gene that is upregulated in miMEFs. Suppression of SPARC expression in miMEFs using small interference RNA (siRNA) displayed gradual detachment of miMEFs. Furthermore, we found a significant reduction of OCT4- and SSEA3-positive hPS cell population maintained on SPARC siRNA-miMEFs compared to on miMEFs by flow cytometrical analysis. These findings suggest that SPARC plays a critical role in the maintenance of miMEFs without loss of cell number and might be a key component for supporting the culture of hPSCs.