Myo10 is an unconventional myosin that localizes to and induces filopodia, structures that are critical for growing axons. In addition to the ~240-kDa full-length Myo10, brain expresses a ~165 kDa isoform that lacks a functional motor domain and is known as headless Myo10. We and others have hypothesized that headless Myo10 acts as an endogenous dominant negative of full-length Myo10, but this hypothesis has not been tested, and the function of headless Myo10 remains unknown. We find that cortical neurons express both headless and full-length Myo10 and report the first isoform-specific localization of Myo10 in brain, which shows enrichment of headless Myo10 in regions of proliferating and migrating cells, including the embryonic ventricular zone and the postnatal rostral migratory stream. We also find that headless and full-length Myo10 are expressed in embryonic and neuronal stem cells. To directly test the function of headless and full-length Myo10, we used RNAi specific to each isoform in mouse cortical neuron cultures. Knockdown of full-length Myo10 reduces axon outgrowth, whereas knockdown of headless Myo10 increases axon outgrowth. To test whether headless Myo10 antagonizes full-length Myo10, we coexpressed both isoforms in COS-7 cells, which revealed that headless Myo10 suppresses the filopodia-inducing activity of full-length Myo10. Together, these results demonstrate that headless Myo10 can function as a negative regulator of full-length Myo10 and that the two isoforms of Myo10 have opposing roles in axon outgrowth.