The photo-induced ring-closure reaction from trans-2-hydroxychalcone (Ct) to flav-3-en-2-ol (F) in acetonitrile was studied by time-resolved infrared (TR-IR) spectroscopy. TR-IR difference spectra observed immediately after photoexcitation of Ct contained negative peaks at 1604 and 1664 cm(-1) where Ct has vibrational bands. Their amplitudes became more negative with a common time constant of 10 ms, which indicates that intermediate species generated within the time-resolution also had vibrational bands at the wavenumbers. We analyzed the observed TR-IR difference spectra assuming that the spectra consist of three spectral components due to Ct, F, and an intermediate and extracted the vibrational spectrum of the intermediate by subtracting contributions of Ct and F from the observed difference spectra. Based on vibrational analysis by density functional theory calculations, the intermediate of the photo-ring-closure reaction was assigned to cis-2-hydroxychalcone hydrogen-bonded to the solvent molecules.