SummaryArf GTPases are known to be key regulators of vesicle budding in various steps of membrane traffic in yeast and animal cells. We cloned the Arabidopsis Arf1 homologue, AtArf1, and examined its function. AtArf1 complements yeast arf1 arf2 mutants and its GFP‐fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The expression of dominant negative mutants of AtArf1 in tobacco and Arabidopsis cultured cells affected the localization of co‐expressed GFP‐tagged proteins in a variety of ways. AtArf1 Q71L and AtArf1 T31N, GTP‐ and GDP‐fixed mutants, respectively, changed the localization of a cis‐Golgi marker, AtErd2‐GFP, from the Golgi apparatus to the endoplasmic reticulum but not that of GFP‐AtRer1B or GFP‐AtSed5. GFP‐AtRer1B and GFP‐AtSed5 were accumulated in aberrant structures of the Golgi by AtArf1 Q71L. A soluble vacuolar protein, sporamin‐GFP, was also located to the ER by AtArf1 Q71L. These results indicate that AtArf1 play roles in the vesicular transport between the ER and the Golgi and in the maintenance of the normal Golgi organization in plant cells.