ACAP3 belongs to ACAP family of GTPase-activating proteins (GAPs) for the small GTPase ADP-ribosylation factor (Arf). However, its specificity to Arf isoforms and physiological functions remain unclear. Here, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild type of ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild type, GTP- or GDP-locked mutant of Arf6. Thus, cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine nucleotide exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.