Functional repair of tendons remains a challenge to be overcame for both clinicians and scientists. We have previously reported a three-dimensional (3D) RADA nanofiber hydrogel that provides a suitable microenvironment for human tendon stem/progenitor cells (TSPCs) survival and tenogenesis. In the current study, we explore the potential of in vivo patellar tendon repair by human TSPC-laden RADA hydrogel in rats, which were sacrificed at 4 and 8 weeks after operation. Hind limb function test, macroscopical and histological examination, tendon cell amount and alignment analysis, and radiographic assessments were performed at several time points. Our results demonstrated that human TSPC-laden RADA hydrogel (RADA+TSPC group) boosted in vivo patellar tendon repair with better ambulatory function recovery compared to the control groups, in which tendon defects were untreated (Defect group) or treated with RADA hydrogel alone (RADA group). In addition, better macroscopic appearance and improved matrix organization in the repaired tendon with less cell amount and reduced adipocyte accumulation and blood vessel formation were observed in the RADA+TSPC group. Moreover, tendon defect treated with TSPC-laden RADA hydrogel resulted in diminished heterotopic ossification (HO) at 8weeks post-operation, which indicated by both X-ray examination and micro-CT scan. Taken together, the combination of TSPC and nanofiber hydrogel provide an optimistic alternative method to accelerate functional tendon repair with reduced heterotopic ossification.