The polyamine (PA) metabolism is involved in cell proliferation and differentiation. Increased cellular PA levels are observed in different types of cancers. Products of PA oxidation induce apoptosis in cancer cells. These observations open a perspective to exploit the enzymes of PA catabolism as a target for anticancer drug design. The substances capable to enhance PA oxidation may become potential anticancer agents. The goal of our study was to explore how the mode of ligand binding with a PA catabolic enzyme is associated with its stimulatory or inhibitory effect upon PA oxidation. Murine N1-acetylpolyamine oxidase (5LFO) crystalline structure was used for molecular docking with ligands of various chemical structures. In vitro experiments were carried out to evaluate the action of the tested compounds upon PA oxidative deamination in a cell-free test system from rat liver. Two amino acid residues (Aps211 and Tyr204) in the structure of 5LFO were found to be significant for binding with the tested compounds. 19 out of 51 screened compounds were activators and 17 were inhibitors of oxidative deamination of PA. Taken together, these results enabled to construct a recognition model with characteristic descriptors depicting activators and inhibitors. The general tendency indicated that a strong interaction with Asp211 or Tyr204 was rather typical for activators. The understanding of how the structure determines the binding mode of compounds with PA catabolic enzyme may help in explanation of their structure-activity relationship and thus promote structure-based drug design.