The diffusion of acetonitrile in conformational isomers of the aliphatic polyurethane, H12MDI (4,4＇-dicyclohexylmethane diisocyanate)/BD (1,4-butanediol)/PTMO (poly(tetramethylene oxide)), was investigated at a fixed hard segment content of 29.9 wt%. The effective diffusion coefficient, measured experimentally using FTIR-ATR (Fourier transform infrared-attenuated total reflectance) spectroscopy, decreased as the trans-trans percentage in the hard segment increased. The spectra for the polyurethanes revealed higher fractions of hydrogen-bound C=O (carbonyl) groups at higher trans-trans percentages, which was consistent with higher values of hard segment T-g. During acetonitrile diffusion experiments, a shift from hydrogen-bound to free carbonyl groups in the hard segment domains occurred and hydrogen-bound C=N and NH peaks appeared suggesting that acetonitrile is solvating to the hard segments in the polymer. Based on these findings, the trend observed for the effective diffusion coefficient may be attributed to tortuosity and penetrant solvation in the polyurethane.