Ultrasonic backscatter techniques are being developed to detect changes in cancellous bone caused by osteoporosis. One technique, called the backscatter difference technique, measures the power difference between two portions of a backscatter signal. The goal of the present study is to investigate how bone mineral density (BMD) and the microstructure of human cancellous bone influence four backscatter difference parameters: the normalized mean of the backscatter difference (nMBD) spectrum, the normalized slope of the backscatter difference spectrum, the normalized intercept of the backscatter difference spectrum, and the normalized backscatter amplitude ratio (nBAR). Ultrasonic measurements were performed with a 3.5 MHz broadband transducer on 54 specimens of human cancellous bone from the proximal femur. Volumetric BMD and the microstructural characteristics of the specimens were measured using x-ray micro-computed tomography. Of the four ultrasonic parameters studied, nMBD and nBAR demonstrated the strongest univariate correlations with density and microstructure. Multivariate analyses indicated that nMBD and nBAR depended on trabecular separation and possibly other microstructural characteristics of the specimens independently of BMD. These findings suggest that nMBD and nBAR may be sensitive to changes in the density and microstructure of bone caused by osteoporosis.