Mammary epithelial cell (MEC) number is an important determinant of milk production in lactating dairy cows. IGF-I increases IGF binding protein-3 (IGFBP-3) production in these cells, which plays a role in its ability to enhance proliferation. In the present study, we show that the apoptotic factor anisomycin (ANS) also increases IGFBP-3 mRNA and protein in a dose- and concentration-dependent manner that mirrors activation of caspase-3 and -7, with significant increases in both IGFBP-3 protein and caspase activation observed by 3 h. Knock-down of IGFBP-3 with small interfering (si) RNA attenuated the ability of ANS to induce apoptosis, while knock-down of IGFBP-2, the other major IGFBP made by bovine MEC, had no effect. Reducing IGFBP-3 also decreased the ability of ANS to induce mitochondrial cytochrome c release, indicating its involvement in the intrinsic apoptotic pathway. In contrast, transfection with IGFBP-3 in the absence of ANS failed to induce apoptosis. Since both the mitogen IGF-I and the apoptotic inducer ANS increase IGFBP-3 production in MEC, we proposed that cellular localization might determine IGFBP-3 action. While both IGF-I and ANS stimulated the release of IGFBP-3 into conditioned media, only ANS induced nuclear localization of IGFBP-3. A pan-caspase inhibitor had no effect on ANS-induced nuclear localization of IGFBP-3, indicating that nuclear entry of IGFBP-3 precedes caspase activation. Treatment with IGF-I had no effect on ANS-induced nuclear localization, but did block ANS-induced apoptosis. In summary, our data indicate that IGFBP-3 plays a role in stress-induced apoptosis that may require nuclear localization in non-transformed MEC. J. Cell. Physiol. 228: 734–742, 2013. © 2012 Wiley Periodicals, Inc.