The femtosecond degenerate four-wave mixing (fs-DFWM) technique is applied for the measurement of accurate rotational constants of cyclobutane (C4H8). The vibrational levels of C4H8 exhibit tunneling splitting due to the ring-puckering interconversion between the symmetry-equivalent D-2d minima via a planar D-4h barrier. For the nu = 0 ground state, the fs-DFWM method yields a rotational constant B-0(+) = 10663.452(18) MHz. The ring-puckering tunneling leads to slightly different rotational constants for the 0(+) and 0(-) levels, B-0(+)-B-0(-)=33 +/- 2 kHz. This difference increases by a factor of similar to 90 in the v = 1(+)/1(-) ring-puckering states to B-1(+) -B-1(-)=-3059 +/- 4 kHz. Combining the experimental rotational constants with the structure parameters and rotational constants calculated by high-level ab initio calculations allows us to determine accurate equilibrium and vibrationally averaged structure parameters for cyclobutane, for example, r(e)(C-C) = 1.5474 angstrom, r(e)(C-H-axial) = 1.0830 angstrom, r,(C-H-equatorial) = 1.0810 angstrom, and ring puckering angle theta(e) = 29.8 degrees.