The microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pull-down assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minus-end anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosome-related mechanisms and support the idea that different EB1-EB3 dimer species have distinct functions within cells.