We measure in vitro tissue birefringence in the liver of hypercholesterolemic rats with polarization-sensitive optical coherence tomography. Tissue birefringence is determined by measuring the phase retardation as a function of tissue depth. The birefringence of such a sample is usually due to the narrow fibrous structures that cannot be resolved by a standard optical coherence tomography system. Anisotropic structures are formed in the hypercholesterolemic rat liver, which is quite different from the isotropic nature of healthy liver. Birefringence is evaluated to give an order of magnitude of 4.48 ×10-4 at 790 nm in hypercholesterolemic rat liver. The infiltration of macrophages and increased collagen deposition should be major causes for tissue birefringence in hypercholesterolemic liver.