Axon regeneration in the central nervous system is inefficient. However, the neurons in the peripheral nervous system display robust regeneration after injury, indicating that axonal regeneration is differentially controlled under various conditions. To identify those molecules regulating axon regeneration, comparative analysis from dorsal root ganglion neurons at embryonic or adult stages is utilized, which reveals that PDK1 is functions as a negative regulator of axon regeneration. PDK1 is downregulated in embryonic neurons after axotomy. In contrast, sciatic nerve axotomy upregulated PDK1 at protein levels from adult mice. The knockdown of PDK1 or the chemical inhibition of PDK1 promotes axon regeneration in vitro and in vivo. Here we present PDK1 as a new player to negatively regulate axon regeneration and as a potential target in the development of therapeutic applications.