Ultrasonic backscatter techniques may offer a useful approach for detecting changes in bone caused by osteoporosis. The goal of this study was to investigate how bone mineral density (BMD) and the microstructure of human cancellous bone affect three ultrasonic backscatter parameters that have been identified as potentially useful for ultrasonic bone assessment purposes: the apparent integrated backscatter (AIB), the frequency slope of apparent backscatter (FSAB), and the frequency intercept of apparent backscatter (FIAB). Ultrasonic measurements were performed with a 3.5-MHz broadband transducer on 54 specimens of human cancellous bone prepared from the proximal femur. Microstructural parameters and BMD were measured using X-ray microcomputed tomography (micro-CT). Relationships between AIB, FSAB, FIAB, and the micro-CT parameters were investigated using univariate and multivariate statistical analysis techniques. Moderate-to-strong univariate correlations were observed between the backscatter parameters and microstructure and BMD in many cases. The partial correlation analysis indicated that the backscatter parameters are dependent on microstructure independently of BMD in some cases. Multiple stepwise linear regression analysis used to generate multivariate models found that microstructure was a significant predictor of the backscatter parameters in most cases.