Microtubule reorganization is necessary for many cellular functions such as cell migration, cell polarity and cell division. Dynamin was originally identified as a microtubule-binding protein. Previous limited digestion experiment revealed that C-terminal 100-amino acids proline rich domain (PRD) of dynamin is responsible for microtubule binding in vitro. However, as obvious localization of dynamin along microtubules is only observed at the spindle midzone during mitosis but not in interphase cells, it remains unclear how dynamin interacts with microtubules in vivo. Here, we report that GFP-dynamin-2-(1-786), a truncated mutant lacking a C-terminal portion of the PRD, localized along microtubules in interphase HeLa cells. GFP-dynamin-2-wild type (WT) and GFP-dynamin-2-(1-745), a construct that was further truncated to remove the entire PRD, localized in discrete punctate structures but not along microtubules. These data suggest that the N-terminal (residues 746-786) but not the entire PRD is necessary for the interaction of dynamin-2 with microtubules in the cell and that the C-terminus of PRD (787-870) negatively regulate this interaction. Microtubules in cells expressing GFP-dynamin-2-(1-786) were stabilized against exposure to cold. These results provide a first evidence for a regulated interaction of dynamin-2 with microtubules in cultured mammalian cells.