Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.