One of the major barriers to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that originate from the myelin sheath and glial scar. So far, only a small number of pharmacological compounds have exhibited functional activity against CNS inhibitors in promoting axon regeneration after injury. To search for novel compounds that enhance neurite outgrowth in vitro, we initiated a screen of a collection of natural products. We identified four compounds with the potential to promote growth over a myelin substrate. Of these, Amphotericin B (AmB) was shown to enhance neurite outgrowth and antagonize activities of major myelin associated inhibitors and glial-scar-derived chondroitin sulfate proteoglycans. AmB was found to activate Akt and thereby suppress the activity of glycogen synthase kinase 3 beta. Also, a cell permeable peptide that inhibits Akt activity was shown to block the effect of AmB in promoting axonal growth, while another peptide that increases Akt activity stimulated axonal growth in the presence of the myelin associated inhibitors. Our results suggest that AmB can promote neurite outgrowth over a wide range of inhibitory substrates via a mechanism that involves activation of Akt.