Emerging evidence demonstrates that RUNX3 is a tumor suppressor in breast cancer. Inactivation of RUNX3 in mice results in spontaneous mammary gland tumors, and decreased or silenced expression of RUNX3 is frequently found in breast cancer cell lines and human breast cancer samples. However, the underlying mechanism for initiating RUNX3 inactivation in breast cancer remains elusive. Here, we identify prolyl isomerase Pinl, which is often overexpressed in breast cancer, as a key regulator of RUNX3 inactivation. In human breast cancer cell lines and breast cancer samples, expression of Pinl inversely correlates with the expression of RUNX3. In addition, Pinl recognizes four phosphorylated Ser/Thr-Pro motifs in RUNX3 via its WW domain. Binding of Pinl to RUNX3 suppresses the transcriptional activity of RUNX3. Furthermore, Pinl reduces the cellular levels of RUNX3 in an isomerase activity-dependent manner by inducing the ubiquitination and proteasomal degradation of RUNX3. Knocking down Pinl enhances the cellular levels and transcriptional activity of RUNX3 by inhibiting the ubiquitination and degradation of RUNX3. Our results identify Pinl as a new regulator of RUNX3 inactivation in breast cancer.