Pluripotent tendon cells (PTCs) play important roles in maintaining metabolic balance and the development of tendinopathy. Recently, substance P (SP) was identified as an important factor in the pathogenesis of tendinopathy. A better understanding of SP's effects on PTCs is essential to gain more insight into tendinopathy. This study aims to investigate the effects of SP on PTCs in vitro and in vivo. For the in vitro study, we quantified exogenous SP production by PTCs and determined the effects of SP on the proliferation and differentiation of PTCs. For the in vivo study, we used a rat patella tendon injection model to investigate the biological effects of SP on PTCs and tendons. Our results showed that exogenous SP production by PTCs was 1.80 pg/10(6) cells and SP significantly enhanced the proliferation of PTCs. Reverse transcription polymerase chain reaction (RT-PCR) results showed that SP upregulated the expression of non-tenocyte genes but downregulated the expression of tenocyte-related genes in the differentiation of PTCs. Moreover, Histological examination showed that SP induced disorganizes of tissue and tendinosis-like changes in vivo. Taken together, these findings indicate that SP enhanced PTCs' proliferation, induced non-tenocyte differentiation and plays an important role in the development of tendinopathy.