ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b 1 disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To 2 determine whether ARL13B is required within cilia, we knocked in a cilia-excluded 3 variant of ARL13B (V358A) and showed it retains all known biochemical function. We 4 found that ARL13B V358A protein was expressed but could not be detected in cilia, even 5 when retrograde ciliary transport was blocked. We showed Arl13b V358A/V358A mice are 6 viable and fertile with normal Shh signal transduction. However, in contrast to wild type 7 cilia, Arl13b V358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. 8These data indicate that ARL13B's role within cilia can be uncoupled from its function 9 outside of cilia. Furthermore, these data imply that the cilia defects upon complete 10 absence of ARL13B do not underlie the alterations in Shh transduction, which is 11 unexpected given the requirement of cilia for Shh transduction. 12