Mesenchymal stem cells (MSCs), first described in human bone marrow, are emerging as promising cell-based therapeutics for a wide range of diseases (Caplan and Correa, 2011). MSCs have been isolated from various organs in the body, and synovial MSCs were first reported by De Bari et al. (2001). We previously reported that synovial MSCs have superior proliferation and chondrogenic potentials as compared to bone marrow-, muscle-, and adipose- derived MSCs in humans (Sakaguchi et al., 2005) and rats (Yoshimura et al., 2007). In addition, administration of synovial MSCs for osteochondral defect promoted cartilage regeneration in a rabbit (Koga et al., 2008) and a pig model (Nakamura et al., 2012). In 2008, we started a clinical trial in human and obtained satisfactory results of symptoms and regenerated cartilage by Magnetic Resonance Imaging (Sekiya et al., 2015). We have also engaged in multiple research lines using synovial MSCs for meniscus regeneration in rats (Horie et al., 2009; Horie et al., 2012; Katagiri et al., 2013; Okuno et al., 2014; Ozeki et al., 2015). In this article, we demonstrated how to harvest the synovium including infrapatellar fat pad from a rat knee joint, and to describe the technique of isolation and culture of rat synovial MSCs.