Primary human hepatocytes are widely used for basic research, pharmaceutical testing, and therapeutic concepts in regenerative medicine. Human hepatocytes can be isolated from resected liver tissue. Preoperative portal vein embolization (PVE) is increasingly used to decrease the risk of delayed postoperative liver regeneration by induction of selective hypertrophy of the future remnant liver tissue. The aim of this study was to investigate the effect of PVE on the outcome of hepatocyte isolation. Primary human hepatocytes were isolated from liver tissue obtained from partial hepatectomies (n = 190) using the two-step collagenase perfusion technique followed by Percoll purification. Of these hepatectomies, 27 isolations (14.2%) were performed using liver tissue obtained from patients undergoing PVE before surgery. All isolations were characterized using parameters that had been described in the literature as relevant for the outcome of hepatocyte isolation. The isolation outcomes of the PVE and the non-PVE groups were then compared before and after Percoll purification. Metabolic parameters (transaminases, urea, albumin, and vascular endothelial growth factor secretion) were measured in the supernatant of cultured hepatocytes for more than 6 days (PVE: n = 4 and non-PVE: n = 3). The PVE and non-PVE groups were similar in regard to donor parameters (sex, age, and indication for surgery), isolation parameters (liver weight and cold ischemia time), and the quality of the liver tissue. The mean initial viable cell yield did not differ between the PVE and non-PVE groups (10.16 ± 2.03 × 10(6) cells/g vs. 9.70 ± 0.73 × 10(6) cells/g, p = 0.499). The initial viability was slightly better in the PVE group (77.8% ± 2.03% vs. 74.4% ± 1.06%). The mean viable cell yield (p = 0.819) and the mean viability (p = 0.141) after Percoll purification did not differ between the groups. PVE had no effect on enzyme leakage and metabolic activity of cultured hepatocytes. Although PVE leads to drastic metabolic alterations and changes in hepatic blood flow, embolized liver tissue is a suitable source for the isolation of primary human hepatocytes and is equivalent to untreated liver tissue in regard to cell yield and viability.