Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az₁) and antizyme inhibitor (AzIN). Az₁ suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az₁ binding. The structural basis of the Az₁-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az₁ complexed with either ODC or AzIN. Structural analysis revealed that Az₁ sterically blocks ODC homodimerization. Moreover, Az₁ binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az₁ for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az₁-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN–Az₁ structure suggests how AzIN may effectively compete with ODC for Az₁ to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.