We investigated the excited state intermolecular proton transfer of pyrene-urea compounds, 1PUP and 4PUP, followed by a tautomer formation in the presence of an acetate ion. The drastic red-shift and enhancement of fluorescence intensity of tautomer of 4PUP were obtained in comparison to 1PUP. The kinetics of formation and decay of excited state tautomer was revealed spectroscopically. Our findings will contribute to the molecular design of fluorescence materials with pyrene moieties based on the substitution patterns.