Ultrasound elastography is a method that can be used to determine the elastic properties of soft tissues and it has been recently applied to study of articular cartilage. While ultrasound elastography techniques assume a constant ultrasound speed in tissue under mechanical compression, ultrasound speed in articular cartilage has been found to vary depending on the loading conditions. This may limit the quantitative use of the technique for determination of the elastic properties of articular cartilage along the axis of ultrasound propagation. The aim of the present study was to investigate the origin of the load-related variation in ultrasound speed. Samples of human and bovine articular cartilage (n = 82) were mechanically and acoustically tested during unconfined compression. A statistically significant (p < 0.05) variation of ultrasound speed was found in cartilage during a stress-relaxation test. A finite element model was constructed by exploiting microscopically determined collagen and proteoglycan contents, collagen orientation and biochemical analyses of water content. From the finite element simulations, collagen orientation and the void ratio (fluid-to-solid ratio) as a function of time were assessed and, together with the experimentally determined ultrasound speed, a linear model predicting variation of the ultrasound speed in human articular cartilage under mechanical compression was established. The model predicted compression-related ultrasound speed with an error of <0.3% at each time point. The effect of strain rate on variation of ultrasound speed was tested in bovine cartilage samples. The decrease in ultrasound speed was found to be proportional to the strain rate. The results suggest that ultrasound speed in articular cartilage is controlled mainly by collagen orientation and the void ratio and depends on the imposed strain rate. A numerical simulation revealed that the compression-related decrease in ultrasound speed induces notable errors in mechano-acoustically determined strain. A method to eliminate the compression-related errors in measured strain and elastic properties may be needed in mechano-acoustic measurements of articular cartilage.