Important pathophysiological characteristics of human tumors are the presence of hypoxia and of an extracellular acidosis. Both metabolic parameters can affect the malignant behavior of tumors but also the response to treatments. Here the modulation of the ERK1/2 signaling plays a relevant role. ERK1/2 activation is controlled by the dual-specificity phosphatase 6 (Dusp6) and by this modulates cellular processes. For this reason, the study analyzed the impact of hypoxia and/or extracellular acidosis on the expression of Dusp6. Several tumor cell lines were exposed to control conditions (room air, pH = 7.4), hypoxia (pO2 = 1.5 mmHg), and/or an extracellular pH of 6.6 for 24 h. Gene expression was measured by qPCR, RNA sequencing, and Western blot. In addition, the expression and activation of signaling molecules (ERK1/2, mTOR) were analyzed. Most of the tumor lines showed a significant reduction in the Dusp6 expression during acidosis and hypoxia. The combination of hypoxia+acidosis simultaneously had the strongest impact on Dusp6 mRNA expression. The ERK1/2 phosphorylation showed an inverse behavior and was increased during acidosis and hypoxia. Therefore, the ERK1/2 activation might result (at least partially) from reduced phosphatase activity. ROS generation during acidosis was not responsible for Dusp6 changes. However, mTor activation correlated well with Dusp6 expression. Additionally, other regulating proteins, like the transcription factor Ets2 and the p53, which were regulated by hypoxia and acidosis as well, correlated strongly with Dusp6 expression. It can be concluded that ERK1/2 activation during acidosis and/or hypoxia results from Dusp6 repression, which could be induced by mTor signaling or the expression of other regulating proteins.