Cell survival, homeostasis and cell polarity rely on the control of membrane trafficking pathways. The RUN domain (comprised of the RPIP8, UNC-14, and NESCA proteins) has been suggested to be implicated in small GTPase-mediated membrane trafficking and cell polarity. Accumulating evidence supports the hypothesis that the RUN domain-containing proteins might be responsible for an interaction with a filamentous network linked to actin cytoskeleton and/or microtubules. In addition, several downstream molecules of PI3K are involved in regulation of the membrane trafficking by interacting with vesicle-associated RUN proteins such as RUFY family proteins. In this review, we summarize the background of RUN domain research with an emphasis on the interaction between RUN domain proteins including RUFY proteins (designated as RUN and FYVE domain-containing proteins) and several small GTPases with respect to the regulation of cell polarity and membrane trafficking on filamentous network.