<b>Introduction:</b> UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in <i>C. elegans</i>. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance. <b>Methods:</b> Growth cone imaging in mutants, combined with Cas9 genome editing and genetic analysis, were used to analyze the role of a novel short isoform on <i>
unc-5</i> in growth cone polarity and protrusion. <b>Results:</b> Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the <i>
unc-5</i> long isoforms were hypomorphic, suggesting a role of <i>
unc-5B</i> short. A mutation specifically affecting <i>
unc-5B</i> caused loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of <i>
unc-5</i> long mutations. Transgenic expression of <i>
unc-5B</i> partially rescued <i>
unc-5</i> axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with <i>
unc-40</i> and <i>
unc-6</i> suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion. <b>Discussion:</b> These results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion.