Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipes L-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8 U/ml) have T-1/2 19.8 h, elimination constant 0.088 U/h and apparent volume distribution 85 U/ml. Also, Af-METase has two maxima one at A(280nm) (apo-enzyme) and at A(420nm) (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15 min then the absorbance at 420 nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A(280/420) ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30 h. Rabbits plasma methionine was depleted from 18.7 mu M (control) to 8.8 mu M after 1 h of enzyme injection and completely omitted after 2 h till 19 h, assuming the sustainability of negligible levels of methionine (<2 mu M) in plasma of rabbits, for about 17 h. Upon infusion of PLP, the T-1/2 of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33 h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4 h of its infusion, the A(280/420) ratio being not relatively changed till 45 h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC50 0.001 U/ml, 0.26 U/ml and 0.37 U/ml, respectively. Published by Elsevier Inc.