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Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue‐engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self‐renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic‐specific markers and an increasing COL1A1:3A1 ratio. The pSC‐derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic‐co‐glycolic) acid scaffolds. Finally, engineered tendon‐like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte‐scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration. Stem Cells Translational Medicine 2017;6:196–208