Acetogenins are among the most potent of the known inhibitors of complex I (NADH-ubiquinone oxidoreductase) in mitochondrial electron transfer system. Elucidation of the dynamic function of the alkyl spacer linking the two toxophores (i.e., the hydroxylated tetrahydrofuran and the γ-lactone rings) is critical for fully understanding their inhibition mechanism. To this end, using molecular dynamics simulations a structure-activity relationship study of a series of acetogenins was performed for the first time using this approach. Our results clearly indicated that both, the length and the molecular flexibility of the spacer, were crucial for taking an active conformation. A partially folded conformation with an optimal length (bis-tetrahydrofuran rings and 13 carbon atoms) of about 16 Å with a high molecular flexibility might depict an active form of the spacer. In addition, we demonstrated that the bis-tetrahydrofuran derivatives are able to overcome the shortage of the length of the spacer more efficiently than the mono-tetrahydrofuran derivatives with the help of the additional tetrahydrofuran, which acts as a pseudospacer. Our results obtained from molecular dynamics calculations supported the use of a combined decane/water system as a good solvent model to simulate the biological environment of acetogenins acting as inhibitor of complex I.