To analyze the role of PAI-1 (plasminogen activator inhibitor 1) in endometriotic lesion growth, we studied the effect of PAI-1 inhibition by PAI-039 using a homologous mouse model of endometriosis that allows noninvasive monitoring. Endometrial tissue from donor mice was collected, labeled with mCherry adenovirus, and implanted into a subcutaneous pocket on the ventral abdomen of recipient mice. Seven days after transplantation, mice were randomly allocated in two groups and treated once daily for 2 weeks with either vehicle (control group) or PAI-1 inhibitor (PAI-039 group). Endometriotic lesion size generated in recipient mice was monitored by mCherry signal. Animals were euthanized 21 days after endometrial tissue implantation and endometriotic lesions were harvested for fibrin deposit and vascularization analyses. Collagen content was also examined to determine the overall effects of proteolysis on extracellular matrix degradation. We demonstrated that endometriotic lesions generated in recipient mice from both groups presented characteristics typical of human endometriotic lesions. We observed a significant decrease in fluorescence signal in endometriotic lesions from the PAI-039 group at the beginning of the treatment correlated with a decrease in endometriotic lesion size. PAI-1 inhibition significantly decreased lesion cell proliferation. In addition, endometriotic lesions from the PAI-1 inhibition group showed a decreased percentage of neovascularization as well as fibrin deposits. However, the density and distribution of collagen were not affected by PAI-039. Our results suggest that in vivo inhibition of PAI-1 by PAI-039 may be a useful strategy to reduce endometriotic lesion size by blocking angiogenesis.