An accurate gas-phase structure determination of cis-hex-3-ene-1,5-diyne (1), the parent molecule for the Bergman cyclization reaction, has been achieved through a combination of Fourier transform microwave spectroscopy and computational quantum chemistry. The microwave spectra of seven isotopes of enediyne 1 have been observed using a pulsed-nozzle Fourier transform spectrometer. The ground-state rotational constants were corrected for vibrational effects and the equilibrium structure (R-e) was deduced. The equilibrium structure displays excellent agreement with that obtained from high-level ab initio calculations (CCSD(T)/cc-pVTZ). Both approaches confirm that the molecule has C-2v symmetry with slight deviations of the alkyne units (C-C drop C-H) from linearity. These groups distort away from each other in contra-distinction to their distortion during the Bergman cyclization reaction. The C1-C6 distance (4.32 Angstrom), which has been viewed as a critical parameter governing reactivity in the Bergman cyclization, is notably longer than an earlier, widely publicized value (4.12 Angstrom). An important role of the macrocyclic ring in enediyne antibiotics is to overcome an intrinsic distortion of the enediyne moiety in the direction opposite that of the reaction coordinate for Bergman cyclization.