Mechanisms of transmesothelial invasion of ovarian cancer are still poorly understood. Here we examined whether this phenomenon may be determined by an expression of intercellular junctions in peritoneal mesothelial cells (PMCs). Analysis of ovarian tumors showed that cancer cells are localized below an intact layer of PMCs. The PMCs located near the invaded cancer cells displayed low expression of connexin 43, E-cadherin, occludin, and desmoglein, as well as expressed SA-β-Gal, a marker of senescence. Experiments in vitro showed that senescent PMCs exhibited decreased levels of the four tested intercellular junctions, and that the invasion of ovarian cancer cells through the PMCs increased proportionally to the admixture of senescent cells. Intervention studies showed that the expression of connexin 43, E-cadherin, occludin, and desmoglein in senescent PMCs could be restored upon the blockade of p38 MAPK, NF-κB, AKT, JNK, HGF, and TGF-β1. When these molecules were neutralized, the efficiency of the transmesothelial cancer cell invasion was diminished. Collectively, our findings show that the integrity of the peritoneal mesothelium, which is determined by the expression of junctional proteins, is critical for the invasion of ovarian cancer. They also indicate a mechanism by which senescent PMCs may promote the invasive potential of cancer cells.