Neuronal polarization requires coordinated regulation of membrane trafficking and cytoskeletal dynamics. Several signaling proteins are involved in neuronal polarization via modulation of cytoskeletal dynamics in neurites. However, very little is known about signaling proteins in the neuronal soma, which regulate polarized membrane trafficking and neuronal polarization. Protein kinase D (PKD) constitutes a family of serine/threonine-specific protein kinases and is important in regulating Golgi dynamics and membrane trafficking. Here, we show that two members of the PKD family, PKD1 and PKD2, are essential for the establishment and maintenance of neuronal polarity. Loss of function of PKD with inhibitor, dominant negative, and short interfering RNA disrupts polarized membrane trafficking and induces multiple axon formation. Gain of function of PKD can rescue the disruption of polarized membrane trafficking and neuronal polarity caused by cytochalasin D, which results in F-actin depolymerization. PKD1 and PKD2 are also found to be involved in the maintenance of neuronal polarity, as evidenced by the conversion of preexisting dendrites into axons on PKD inhibition. Unlike other polarity proteins, PKD does not interact with the cytoskeleton in neurites. Instead, PKD regulates neuronal polarity through its activity in the Golgi apparatus. These data reveal a novel mechanism regulating neuronal polarity in the Golgi apparatus.