Mitochondrial fission is essential for organelle homeostasis and is driven by the dynamin-related GTPase DRP1, which exists in multiple isoforms generated by alternative splicing. While DRP1 activity is known to be regulated by post-translational modifications, how isoform diversity integrates metabolic signals and membrane context remains poorly understood. In this study, we purified recombinant human DRP1 isoforms and systematically compared their intrinsic GTPase activities under defined biochemical conditions. We show that DRP1 isoforms exhibit distinct enzymatic profiles and display differential sensitivity to inhibition by palmitoyl-CoA, a long-chain fatty acyl-CoA metabolite. In addition, we examined the influence of mitochondrial lipid environments, including cardiolipin and membrane mimetics representative of the outer and inner mitochondrial membranes, on isoform-specific activity. Our findings demonstrate that alternative splicing confers distinct metabolic and lipid responsiveness to DRP1, revealing an additional regulatory layer by which mitochondrial fission may be tuned to cellular metabolic state.