Many biological processes require the co-operative involvement of both microtubules and microfilaments; however, only a few proteins mediating the interaction between microtubules and microfilaments have been identified from plants. In the present study, a cotton kinesin GhKCH2, which contains a CH (calponin homology) domain at the N-terminus, was analysed in vitro and in vivo in order to understand its interaction with the two cytoskeletal elements. A specific antibody against GhKCH2 was prepared and used for immunolabelling experiments. Some GhKCH2 spots appeared along a few microtubules and microfilaments in developing cotton fibres. The His-tagged N-terminus of GhKCH2 (termed GhKCH2-N) could co-precipitate with microfilaments and strongly bind to actin filaments at a ratio of monomeric actin/GhKCH2-N of 1:0.6. The full-length GhKCH2 recombinant protein was shown to bind to and cross-link microtubules and microfilaments in vitro. A GFP-fusion protein GFP-GhKCH2 transiently overexpressed in Arabidopsis protoplasts decorated both microtubules and microfilaments, confirming the binding ability and specificities of GhKCH2 on microtubules and microfilaments in living plant cells. The results of the present study demonstrate that GhKCH2, a plant-specific microtubule-dependent motor protein, not only interacts with microtubules, but also strongly binds to microfilaments. The cytoskeletal dual-binding and cross-linking ability of GhKCH2 may be involved in the interaction between microtubules and microfilaments and the biological processes they co-ordinate together in cotton cells.