ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. EB1 is a microtubule plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing microtubules and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates microtubule nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EBH region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal microtubule nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased microtubule nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and EB1, which is important for microtubule stabilization. Together, our results indicate that Arl4D modulates microtubule nucleation through regulation of the EB1-p150 association at the centrosome. [Media: see text]