The M_r 16,000 NH₂-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor inhibiting endothelial cell function in vitro and neovascularization in vivo. The present study was undertaken to test the ability of 16K hPRL to inhibit the growth of human HCT116 colon cancer cells transplanted s.c. into Rag1^−/− mice. For this purpose, HCT116 cells were stably transfected with an expression vector encoding a peptide that included the signal peptide and first 139 amino acid residues of human prolactin (HCT116^16K). Stable clones of HCT116^16K cells secreted large amounts of biologically active 16K hPRL into the culture medium. Growth of HCT116^16K cells in vitro was not different from wild-type HCT116 (HCT116^wt) or vector-transfected HCT116 (HCT116^vector) cells. Addition of recombinant 16K hPRL had no effect on the proliferation of HCT116^wt cells in vitro. Tumor growth of HCT116^16K cells implanted into Rag1^−/− mice was inhibited 63% in four separate experiments compared with tumors formed from HCT116^wt or HCT116^vector cells. Inhibition of tumor growth of HCT116^16K cells was correlated with a decrease in microvascular density by 44%. These data demonstrate that biologically active 16K hPRL can be expressed and secreted from human colon cancer cells using a gene transfer approach and that production of 16K hPRL by these cells was capable of inhibiting tumor growth and neovascularization. These findings support the potential of 16K hPRL as a therapeutic agent for the treatment of colorectal cancer.