Photoactivation in the Photoactive Yellow Protein, a bacterial blue light photoreceptor, proceeds via photo-isomerization of the double C=C bond in the covalently attached chromophore. Quantum chemistry calculations, however, have suggested that in addition to double bond photo-isomerization, the isolated chromophore and many of its analogues, can isomerize around a single C-C bond as well. Whereas double bond photo-isomerization has been observed with x-ray crystallography, experimental evidence for single bond photo-isomerization is currently lacking. Therefore, we have synthesized a chromophore analogue, in which the formal double bond is covalently locked in a cyclopentenone ring and carried out transient absorption spectroscopy experiments in combination with non-adiabatic molecular dynamics simulations to reveal that the locked chromophore isomerizes around the single bond upon photo-activation. Our work thus provides experimental evidence for single bond photo-isomerization in a photoactive yellow protein chromophore analogue and suggests that photo-isomerization is not restricted to the double bonds in conjugated systems. This insight may be useful for designing light-driven molecular switches or motors.