HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.