Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyamine-free media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system.