Small non-coding RNAs (microRNAs) have emerged as important regulators of stress response and thus play a critical role in the development and progression of cancer. They regulate the expression of their target genes on a post-transcriptional level and can act either as tumor suppressors or oncogenes (oncomiRs). MicroRNA expression is regulated by the tumor microenvironment, as for instance shown for hypoxia. However, besides hypoxia also a decrease in extracellular pH (acidosis) is often found in tumors. At present, the impact of acidosis on microRNA expression is widely unknown. Thus, the aim of this study was to analyze the role of tumor acidosis on microRNA expression. The expression was analyzed in two experimental tumor lines, subline AT-1 of the Dunning prostate carcinoma and Walker-256 mammary carcinoma, in vitro and in vivo (subcutaneously implanted in male Copenhagen and Wistar rats, respectively). By Next Generation Sequencing 33 microRNAs were identified in AT-1 and 38 in Walker-256 cells which were regulated by acidosis (pH=6.6 for 24 h). 11 microRNAs were regulated in both tumor cell lines consistently in the same direction. By TaqMan qPCR we could confirm 3 down-regulated (miR-183, miR-203, miR-215) and 1 up-regulated microRNA (miR-7). These results were compared to expression changes under hypoxic condition (pO2=1.5 mmHg, pH=7.4) and combined hypoxia+acidosis (pO2=1.5 mmHg, pH=6.6). Acidosis-sensitive microRNAs were either contrariwise or not regulated by hypoxia. The effect of hypoxia+acidosis resembled an additive effect of acidosis and of hypoxia alone. In vivo most of the microRNAs were downregulated when the tumor pH was artificially lowered by inspiratory hypoxia and metaiodobenzylguanidine (MIBG) administration. In AT-1 tumors microRNAs were mostly regulated in the same direction as in isolated cells whereas in Walker-256 tumors several microRNAs showed an opposite regulation as the cells in vitro. To elucidate the impact of microenvironmental regulation of microRNA expression on cellular behavior, putative microRNA targets were studied. The expression of Brip1, Ercc6l, Ikbke, Per3 and Tlr5 was affected by acidosis. Further studies are needed to clarify whether these changes depended on the altered microRNA expression and which of the regulated microRNAs were critical for target regulation. In conclusion, the regulation of microRNAs differs profoundly depending on whether acidosis, hypoxia or acidosis+hypoxia are present in the tumor. Acidosis regulates microRNAs miR-7, miR-183, miR-203 and miR-215 in prostate and breast cancer cells in vitro and in vivo and could by this influence tumor cell transcriptional program thereby tumor cell behavior.