Motions of the carbonyl group of the Photoactive Yellow Protein (PYP) chromophore have been investigated, for the first time, by femtosecond time-resolved circular dichroism (tr-CD) spectroscopy, in the near-ultraviolet spectral region. The quantitative analysis of the tr-CD signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17−53°.