We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium Rhodopseudomonas palustris, using femtosecond time-resolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH(center dot) (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH(center dot) mediates the signaling-state formation, showing that PapB is the second example of FADH(center dot)-mediated formation of the signaling state after Slr1694 (M. Gauden et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10895-10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH(center dot) was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH(center dot) was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH(center dot) in the photocycle of PapB.