TMEM166 is a novel programmed cell death-related molecule. In this report, we constructed a recombinant adenovirus 5-TMEM166 vector (Ad5-TMEM166) and evaluated its expression and anti-tumor activities in vitro and in vivo. Cell viability analysis revealed that the adenovirus-mediated increase of TMEM166 inhibited tumor cell growth in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy (via inhibition of mTOR and activation of p70S6K) and apoptosis (via caspase-3 activation), both of which contributed to cell death and suppression of tumorigenicity. Our data indicated that Ad5-TMEM166 may be a novel gene therapy candidate for cancer.