The microwave spectrum of 4-hexyn-3-ol, CH3—C≡C—CH(OH)—CH2CH3, was recorded in the frequency range of 2–26.5 GHz by molecular jet Fourier transform microwave spectroscopy. The conformational analysis based on quantum chemical calculations yielded nine conformers exhibiting C1 symmetry, of which three could be assigned in the experimental spectrum. The propynyl methyl group CH3—C≡C— experiences internal rotation with a very low barrier due to the presence of the cylindrically symmetric —C≡C— group serving as a spacer to the rest of the molecule, which is 7.161012(7) cm−1, 4.2365(26) cm−1, and 7.9016(39) cm−1 for the three assigned conformers, respectively. The spectrum was analyzed with the program XIAM using the combined axis method and the program BELGI-C1 using the rho axis method and a very flexible Hamiltonian which yields fits with root-mean-square deviations within the measurement accuracy.