The c-Jun N-terminal kinase (JNK) signalling pathway has an established role in cellular stress signalling, cell survival and tumorigenesis. Here, we demonstrate that inhibition of JNK signalling results in partial delocalization of the RNA helicase DDX21 from the nucleolus to the nucleoplasm, increased nucleolar mobility of DDX21 and inhibition of rRNA processing. Furthermore, our results show that JNK signalling regulates DDX21 phosphorylation and protein expression. In conclusion, the results presented in this study reveal a previously unidentified cellular role for JNK signalling in the regulation of nucleolar functions. Based on these results, we propose that JNK-mediated effects on nucleolar homeostasis and rRNA processing should be considered when interpreting cellular phenotypes observed in JNK-deficient cell and animal models.