The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is important in nasopharyngeal carcinoma (NPC) pathogenesis. Activated PI3K and its downstream target Akt are concernful signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. The protein kinase Akt1, one of the Akt family members, is involved in several signaling pathways for tumor development and progression, suggesting that Akt1 might be an interesting target for a molecular tumor therapy. DNAzyme is a single-stranded DNA catalyst that can be engineered to bind to their complementary sequence in the target mRNA and cleave the mRNA. In this study, based on the analysis of sequences, thermodynamics and site distribution within the Akt1 gene, five DNAzymes were designed. We found that the DNAzyme, namely Dz2, strongly inhibited Akt1 mRNA and protein expression in the NPC cell line CNE1-LMP1. It was showed that the inhibition of cell proliferation, stimulation of apoptosis and inhibition of xenograft growth in nude mice can also exerted by Dz2. Besides, the apoptosis was shown to be associated with the suppression of the Bcl-2 and increased expression of Bax. Thus, DNAzyme targeting Akt1, Dz2, can inhibit multiple key tumorigenic processes in vitro and in vivo and may serve as a useful anti-cancer agent in NPC.