Axonal transport is essential for neuronal development and function, and disrupted axonal transport is an important pathophysiological factor in a variety of neurodegenerative diseases. However, the molecular mechanisms that regulate axonal transport, and how disruption of that transport leads to neuronal degeneration, are poorly understood. Here, we report that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) and JNK-associated leucine zipper protein (JLP) are essential for postnatal brain development. Mice with a double knockout (dKO) in Jsap1 and Jlp in the dorsal telencephalon developed progressive neuron loss. Using a primary neuron culture system with induced disruption of targeted genes, combined with gene rescue experiments, we show that JSAP1 and JLP regulate kinesin-1-dependent axonal transport with functional redundancy. We also show that the binding of JSAP1 and JLP to kinesin-1 heavy chain is crucial for interactions between kinesin-1 and microtubules. Furthermore, we describe a molecular mechanism by which defective kinesin-1-dependent axonal transport in Jsap1:Jlp dKO neurons causes axonal degeneration and subsequent neuronal death. JNK hyperactivation due to increased intra-axonal Ca2+ in the Jsap1:Jlp dKO neurons was found to mediate both the axonal degeneration and neuronal death, in cooperation with the Ca2+-dependent protease calpain. Our results indicate that axonal JNK relocates to the nucleus in a dynein-dependent manner, where it activates the transcription factor c-Jun, resulting in neuronal death. Taken together, our data establish JSAP1 and JLP as positive regulators of kinesin-1-dependent axonal transport, which prevents neuronal degeneration.