To investigate the effect of Kim-1 on 786-0 cells in vivo and in vitro, several experiments such as quantitative real-time PCR, Western blot, MTT, colony formation, and flow cytometry were performed to evaluate the biological behavior of 786-0 cells treated with Kim-1 siRNA. Furthermore, the tumor xenograft model was applied to BALB/c nude mice to assess the effect of Kim-1 silencing. Lentivirus-mediated RNAi effectively silenced Kim-1 in 786-0 cells. Kim-1 knockdown significantly inhibited the proliferation and colony formation ability of 786-0 cells (p < 0.01). The cell cycle of 786-0 cells was arrested in the G0/G1 phase (p < 0.01). Early and late apoptosis were significantly increased in the Kim-1 siRNA cells (p < 0.01). In addition, growth of 786-0 cells was significantly inhibited in the Kim-1-silenced mice. In conclusion, knockdown of Kim-1 inhibits the growth of 786-0 cells in vitro and in vivo, indicating that Kim-1 could be used as a potential target for clear cell renal cell carcinoma therapy.