The aim of the present study was to assess the expression of sirtuin (Sirt)1 in tendon stem cells (TSCs) and to elucidate its association with osteogenic and adipogenic differentiation of TSCs. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to detect Sirt1 mRNA and protein levels in TSCs, respectively. TSCs were positive for Sirt1 expression, which was elevated by Sirt1 activator SRT1720 in a time-and concentration-dependent manner, and decreased by Sirt1 inhibitor EX527. TSCs were treated with SRT1720 and EX527 for various time periods and resulting changes in osteogenic and adipogenic protein markers were analyzed using alizarin red and oil red O staining. According to RT-qPCR and western blot analyses, the associated factors beta-catenin, Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 were elevated following increases of Sirt1 levels, while CCAAT/enhancer binding protein (CEBP)alpha and peroxisome proliferator-activated receptor (PPAR)gamma were decreased. These results suggested that osteogenic differentiation capacity was enhanced, while adipogenic differentiation capacity declined. Further mechanistic study revealed that phosphoinositide-3 kinase (PI3K) and AKT were decreased following activation of Sirt1. In conclusion, the present study suggested that Sirt1 promotes the osteogenic differentiation of TSCs through upregulating beta-catenin and Runx2 and inhibits the adipogenic differentiation of TSCs through the PI3K/AKT pathway with downregulation of CEBP alpha and PPAR gamma.