The purpose of the present study is to investigate the effect of cortical bone on acoustic properties of trabecular bone, such as speed of sound (SOS) and normalized broadband ultrasound attenuation (nBUA), in bovine femur in vitro. Twelve trabecular bone samples and three cortical bone plates with thicknesses of 1.00, 1.47, and 2.00 mm were extracted from the proximal end of two bovine femurs. The correlations between acoustic properties and trabecular apparent bone density were also examined before and after attaching a cortical bone plate to the trabecular bone samples. SOS increased linearly with increasing thickness of the cortical plate attached to one side of ultrasonic incidence of the trabecular bone samples, whereas nBUA showed a nonlinear dependence on the thickness of the cortical plate. All the SOS (r = 0.95-0.97) and nBUA (r = 0.53-0.73) measurements with and without the cortical bone plate with various thicknesses were found to exhibit high correlations with the trabecular apparent bone density. These results imply that the acoustic properties measured in the femur with lateral cortical layers in vitro can be useful indices for the prediction of trabecular bone mineral density.