Eg5 is a motor protein of the kinesin family that is critical for spindle assembly during mitosis and has recently been implicated in tumorigenesis. It is largely unknown how Eg5 expression is regulated in cells. In this study, we present the first evidence that cellular Eg5 level is downregulated by Parkin, an E3 ubiquitin ligase well known for its role in the development of Parkinson's disease. Our data show that Parkin does not trigger Eg5 protein degradation through the ubiquitin-proteasome pathway. Instead, Parkin represses Eg5 gene transcription by blocking c-Jun binding to the activator protein 1 site present in the Eg5 promoter. Our data further show that Parkin inactivates the c-Jun NH2-terminal kinase (JNK), resulting in decreased phosphorylation of c-Jun. The inactivation of JNK is further mediated by multiple mono-ubiquitination of Hsp70. Importantly, both the ubiquitination of Hsp70 and the subsequent inactivation of the JNK-c-Jun pathway are crucial for Parkin to downregulate Eg5 expression. These results thus uncover a novel function for Parkin in modulating the expression of Eg5 through the Hsp70-JNK-c-Jun signaling pathway.