Angiopoietin-2 (Angpt2) plays a critical role in angiogenesis and tumour progression. Therapeutic targeting of Angpt2 has been implicated as a promising strategy for cancer treatment. Whereas miRNAs are emerging as important modulators of angiogenesis, regulation of Angpt2 by miRNAs has not been established. Here we firstly report that Ang2 is targeted by a microRNA, miRNA-542-3p, which inhibits tumour progression by impairing Ang2's pro-angiogenic activity. In cultured endothelial cells, miR-542-3p inhibited translation of Angpt2 mRNA by binding to its 3' UTR, and addition of miR-542-3p to cultured endothelial cells attenuated angiogenesis. Administration of miR-542-3p to tumour-bearing mice reduced tumour growth, angiogenesis and metastasis. Furthermore, the level of miR-542-3p in primary breast carcinomas correlated inversely with clinical progression in primary tumour samples from stage III and IV patients. Together, these findings uncover a novel regulatory pathway whereby an anti-angiogenic miR-542-3p directly targets the key angiogenesis-promoting protein Angpt2, suggesting that miR-542-3p may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression.