Renal Cell Carcinoma (RCC) is a higher malignant degree tumor in urinary system. Evidences have suggested that tunicamycin can inhibit many human cancer growth and metastasis. The purpose of this study is to investigate the role of tunicamycin in the progression of RCC cells and RCC mouse model. Results showed that tunicamycin significantly suppressed ACHN and RuCa cells growth, migration and invasion in vitro. We demonstrated that tunicamycin treatment arrested cell cycle of ACHN and RuCa cells. In addition, results indicated that tunicamycin treatment induced apoptosis and down-regulated ERK and AKT expression and phosphorylation levels in ACHN and RuCa cells. Mechanism research showed that overexpression of ERK increased AKT expression and phosphorylation levels and canceled Tunicamycin-down-regulated AKT expression and phosphorylation levels in ACHN and RuCa cells. In addition, overexpression of ERK inhibited Tunicamycin-suppressed growth and aggressiveness of ACHN and RuCa cells. Further, in vivo assays showed that Tunicamycin markedly inhibited tumor growth in ACHN-bearing mouse model via increasing apoptosis. In conclusion, these results indicate that tunicamycin could inhibit growth of RCC cells both in vitro and in vivo via ERK/AKT signal pathway, suggesting tunicamycin may be an efficient anti-cancer agent for RCC therapy.