Previous studies have shown that the tetraspanin CD151 is essential for pathological or physiological angiogenesis. However, the cellular signaling mechanism and the role of the CD151 YRSL sorting motif in in vitro vasculogenesis remains unknown. In this study, the results showed that both CD151 and CD151-ARSA gene delivery were capable of increasing the expression of CD151 at the protein level in human umbilical vein endothelial cells (HUVECs). Moreover, there was no significant difference in CD151 protein expression between the CD151 group and the CD151-ARSA group. Overexpression of CD151 promoted HUVEC cell proliferation, migration and capillary network formation in vitro. However, in the CD151-ARSA group, the abilities of cell proliferation, migration and capillary network formation were all decreased, compared with the CD151 group. Furthermore, the activation of PI3K, Akt and ERK signaling pathways was attenuated in the CD151-ARSA mutant group compared with the CD151 group. This study suggests that the YRSL motif of CD151 plays a key role in CD151-induced angiogenesis. Our observations provide insights into a new mechanism of CD151 regulating angiogenesis via vesicle trafficking.