How EFhd2 may be linked with neurodegenerative diseases is not known, due to the physiological function of EFhd2 being poorly understood. Microtubule associated proteins assist these processes by stabilizing MTs. The MAP tau directly binds and thus stabilizes MT in axons and microtubule associated protein 2 stabilizes MT in dendrites. Defects in synaptic and transport proteins are involved in neurodegenerative diseases by interfering with axonal transport and neural circuit function. Axonal branching during physiological axonal regeneration requires local destabilization of the MT cytoskeleton. This process requires detachment of tau from MTs, which can be mediated by the phosphorylation of tau at many residues. Therefore, controlled tau phosphorylation is a critical physiological process and may be linked with EFhd2. We therefore tested the hypothesis that EFhd2 controls cytoskeletal functions in neurons using EFhd2 knock-out/lacZ knock-in mice. We found that EFhd2 was strongly expressed in the cortex, hippocampus, thalamus and the olfactory bulb. We revealed that EFhd2 is has a negative impact on transport of synaptophysin-GFP containing WZ4002 company vesicles in hippocampal neurons. Specifically, EFhd2 inhibited kinesin mediated microtubule gliding. Taken together, we propose that EFhd2 is a neuronal protein that interferes with kinesin activity. We next asked, whether EFhd2 decreased axonal transport by inhibition of the plus end microtubule motor protein kinesin. We quantified kinesin activity in a microtubule-gliding assay. In this cell free in vitro assay system, purified neuron specific kinesin was coated on a glass surface, overlaid with polymerized MTs and movement of MTs was analyzed by microscopy. Interestingly, the recombinant GST-EFhd2 fusion protein, but not GST alone, inhibited KIF5A mediated MT gliding in a dose-dependent manner. These data indicated that kinesin mediated transport was modulated in the presence of EFhd2. We therefore measured the ATPase activity of KIF5A in the absence or presence of recombinant GST-EFhd2. We did not observe a significant inhibition of kinesin mediated ATPase activity by GST-EFhd2. Thus, EFhd2 slowed kinesin mediated MT gliding, which might be a consequence of reduced kinesin-MT interaction. Strong EFhd2 expression in the brain was detected in the grey matter, including the cortex and hippocampus. Thus, the lacZ expression pattern in EFhd2-gene targeted mice and anti-EFhd2 immunohistochemistry outlined a specific EFhd2 expression in the pyramidal layers of the cortex, in the dentate gyrus, and in the CA1-CA2 regions of the hippocampus. These findings are in line with the in situ hybridizations described in the Allen brain atlas where areas with a high density of neurons also intensively stain for efhd2, whereas regions with fewer neurons also show less efhd2 expression. Brain regions that mostly consist of white matter do not show efhd2 expression. Interestingly, these findings were also confirmed in microarrays from human brain. In the developing embryo, we show for the first time that the EFhd2 protein is expressed in the cortex, hippocampus, and thalamus of E18 brain, indicative of expression of EFhd2 during brain development.