Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease that activates pro-MMP-2 and pro-MMP13, which are related to tumor malignancy. Therefore, probes that specifically image MT1-MMP would be useful for malignant tumor diagnosis. In the present study, we prepared rhodamine X-conjugated anti-MT1- MMP antibody (anti-MT1-MMP mAb-ROX) as an activatable fluorescent probe and evaluated its usefulness for MT1-MMP-specific imaging. Anti-MT1-MMP mAb-ROX was obtained in a quenched form with approximately three ROX molecules per mAb. Its fluorescence intensity increased approximately 14-fold in the presence of detergent, which is suitable for activatable systems. C6 glioma cells and MCF-7 human breast adenocarcinoma cells were used as MT1-MMP-positive and MT1-MMP-negative models, respectively. The fluorescence intensity of C6 cells treated with anti-MT1-MMP mAb-ROX, but not ROX-conjugated isotype control antibody (NC Ab-ROX), increased with time and was significantly higher than that of MCF-7 cells at 6 h (P < 0.001). The fluorescence intensity of cells treated with anti-MT1-MMP mAb-ROX was also suppressed by pre-treatment with a MT1-MMP endocytosis inhibitor (P < 0.05). Furthermore, the probes were intravenously administered to C6 and MCF-7 xenografted mice. The tumor-to-muscle (T/M) ratio of the anti-MT1-MMP mAb-ROX group was 15.1 ± 3.2 at 48 h and was significantly higher than that of the NC Ab-ROX group (T/M ratio = 4.6 ± 3.0, P < 0.05) in C6 xenografted mice, while the T/M ratio of the anti-MT1-MMP mAb-ROX and NC Ab-ROX groups was not different in MCF-7 xenografted mice. These findings suggest that anti-MT1-MMP mAb-ROX is a promising probe for specifically detecting MT1-MMP-expressing tumors.