For understanding the structural features of cytochalasin analogues necessary for hexose transport inhibition, we analyzed the conformations of the inhibitory cytochalasin B (CCB) and cytochalasin A (CCA) and the noninhibitory cytochalasin analogue 21,22-dihydrocytochalasin B (CCBH2) by molecular mechanics computation. Results showed that the 3D-structures of these cytochalasin analogues are regulated by the alpha,beta-unsaturated lactone moiety. Their global minimal conformers differed in the conformation of the lactone moiety. The populations of conformers having the same lactone moiety conformation as that of the global minimal conformer of CCB among the total possible conformers were 93% in CCB, 35% in CCA, and 0% in CCBH2. As these populations were proportional to the magnitudes of the inhibitory effects of these analogues, we conclude that the most stable conformation of CCB with regard to the lactone moiety is the active conformation of cytochalasin analogues for hexose transport inhibition. Thus the conformation of cytochalasin analogues necessary for hexose transport inhibition could be deduced by this novel approach.