Cyclobutenone was characterized by high-resolution Fourier transform microwave spectroscopy for the first time. High-level, first-principles quantum chemical calculations at the B3LYP, CISD, MP2, and CCSD levels of theory were implemented to better understand the molecular structure and obtain model rotational and centrifugal distortion constants to aid in spectral assignment, and the results at the different levels of theory are compared. The assignment of the experimental spectrum provided fits of 2.7 kHz using Watson A-reduced and Watson S-reduced Hamiltonians. No tunneling splittings were observed, suggesting that cyclobutenone is not undergoing ring-puckering tunneling.