Golgi fragmentation is a common feature in multiple neurodegenerative diseases; however, the precise mechanism that causes fragmentation remains obscure. A potential link between Cdk5 and Golgi fragmentation in Alzheimer's disease (AD) was investigated in this study. Because Golgi is physiologically fragmented during mitosis by Cdc2 kinase and current Cdk5-specific chemical inhibitors target Cdc2 as well, development of novel tools to modulate Cdk5 activity was essential. These enzyme modulators, created by fusing TAT sequence to Cdk5 activators and an inhibitor peptide, enable specific activation and inhibition of Cdk5 activity with high temporal control. These genetic tools revealed a major role of Cdk5 in Golgi fragmentation upon beta-amyloid and glutamate stimulation in differentiated neuronal cells and primary neurons. A crucial role of Cdk5 was further confirmed when Cdk5 activation alone resulted in robust Golgi disassembly. The underlying mechanism was unraveled using a chemical genetic screen, which yielded cis-Golgi matrix protein GM130 as a novel substrate of Cdk5. Identification of the Cdk5 phosphorylation site on GM130 suggested a mechanism by which Cdk5 may cause Golgi fragmentation upon deregulation in AD. As Cdk5 is activated in several neurodegenerative diseases where Golgi disassembly also occurs, this may be a common mechanism among multiple disorders.