Aim: To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients. Methods: The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People’s Republic of China (2005 edition, part Ⅲ). Its in vitro and in vivo bioactivities were assayed via proliferation-inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method. Results: The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4×107, 6.7×107, and 3.8×108 U/mg, and the in vivo antitumoral potency was 4.04×107 U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase Ⅰ clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44±91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients. Conclusion: RhEndostatin exhibited a definite proliferation-inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEn-dostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.