ADP-ribosylation factors (Arfs) and Arf-like (Arl) GTPases are key regulators of intracellular vesicle trafficking and Golgi structure. Both Arf and Arl proteins cycle between active GTP-bound and inactive GDP-bound forms, where guanine nucleotide exchange factors (GEFs) regulate the exchange of GDP for GTP, whereas GTPase-activating proteins (GAPs) promote the hydrolysis of bound GTP. Human Arl1 is located at the trans-Golgi network (TGN) and regulates the function and structure of the Golgi complex. However, neither GEFs nor GAPs for human Arl1 have been identified. Here, we report that ArfGAP1, an Arf1 GAP, can promote GTP hydrolysis of Arl1. We show that ArfGAP1 directly interacts with GTP-bound Arl1 and exhibits GAP activity toward Arl1 in vitro. Exogenous expression of ArfGAP1, but not ArfGAP2 and ArfGAP3, causes dissociation of endogenous Arl1 from the TGN. In addition, GAP activity-deficient ArfGAP1 fails to regulate the Golgi localization of Arl1. Using an activity pull-down assay, we demonstrated that ArfGAP1 regulates the levels of Arl1-GTP in cells expressing ArfGAP1-myc or with ArfGAP1 knockdown. Finally, we observed that, similar to expression of putative active Arl1 (Arl1QL), ArfGAP1 knockdown impairs endosome-to-TGN retrograde transport of the Shiga toxin B-subunit. Thus, our findings support the idea that ArfGAP1 acts as an Arl1 GAP to regulate the function of Arl1 in vesicle trafficking at the TGN.