ROPs and Interactor of Constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules which regulate metaxylem secondary cell wall patterning. Yet, loss of function mutants of ICR5 and its closest homologs have not been studied, and hence the function of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5/MIDD1. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of icr2 and icr5 Arabidopsis single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings, however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, while icr2 but not icr5 mutants develop split root hairs demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.