The photoisomerization kinetics of trifloxystrobin (TFS) in acetone under artificial sunlight is reported. HPLC analysis showed the TFS, a strobilurine fungicide of EE conformation, was converted into an equilibrium mixture of four isomers after illumination for 7 h. The isomers were identified as EZ, EE, ZZ, and ZE and were separated in the crystalline form by preparative HPLC and characterized by use of a variety of spectroscopic techniques. The quantum yield and reaction constants for the isomerization reactions were determined. The detailed spectral features of the individual isomers measured by UV, IR, Raman, NMR and mass spectroscopy are presented and compared. The spectra of the isomers were found to be very characteristic, with good analytical significance.