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Human dynamin-like protein 1 (DLP-1) is involved in the fission of mitochondrial outer membranes, a process that helps to maintain mitochondrial morphology and to reduce the accumulation of functional and structural defects in mitochondria. DLP-1 is a ∼80 kDa membrane-interacting protein and contains a GTPase domain, a middle domain, a putative PH-like domain and a GTPase effector domain (GED). While the GED has been suggested to be important on protein oligomerization and GTPase activation, functional relationships between the other domains especially the roles of the middle domain in protein activity remains less clear. In this study, we have investigated the biochemical properties of recombinant DLP-1 wild-type and selected mutants, all expressed in Escherichia coli. The middle domain mutants G350D, R365S and ΔPH (lacking the putative PH-like domain) severely impair the GTPase activity, but have no obvious effects on protein tetramerization and liposome-binding properties, suggesting these mutants probably affect protein intra-molecular interactions. Our study also suggested that proper domain–domain interactions are important for DLP-1 GTPase activity.