Background: MMP-9 is crucial for a normal immune response, but excessive release of this enzyme leads to severe tissue damage. Listeria monocytogenes (LM) is an opportunistic food-borne pathogen causing listerosis, meningitis and sepsis. Heat killed Listeria monocytogenes (HKLM) activates immune system and leads production of cytokines and chemokines. However, nothing is known about the involvement of HKLM in MMP-9 regulation. Therefore we investigated the role of HKLM in the regulation of MMP-9 gene expression in THP-1 cells. Methods: Commercially available heat killed Listeria monocytogenes was used in this study. HKLM-induced MMP-9 expression was assessed with quantitative real-time qPCR and ELISA. Action of HKLM in different signaling pathways were studied by using THP-1-XBlue™ cells (THP-1-cells with NF-κB/AP-1 reporter construct), THP-1-XBlue™-defMyD cells (MyD88-/- THP-1 cells), anti-TLR2 mAb and pharmacological inhibitors. Phospho and total proteins were determined by Western blotting. Results: Increased MMP-9 production (mRNA: 395-Fold; Protein: 8141 pg/ml; P∈<∈0.05) was observed in HKLM stimulated THP-1 cells as compared to the un-stimulated THP-1 cells. This production of MMP-9 was completely abrogated by anti-TLR2 blocking mAb (P∈=∈0.0024). Furthermore, THP-1-XBlue™-defMyD cells were unable to produce MMP-9 in response to HKLM. HKLM- induced activation of NF-kappaB/AP-1 was also observed in THP-1-XBlue™ Cells. In addition, inhibitors of JNK (SP600125), MEK/ERK (U0126; PD98056), p38 MAPK (SB203580) and NF-kappaB (BAY 11-7085, Triptolide and Resveratrol) significantly suppressed (P∈<∈0.05) HKLM-stimulated MMP-9 expression. Conclusion: Our results indicate that HKLM activates TLR2 and NF-κB/AP-1 signaling pathways, leading to up-regulation of MMP-9 production in THP-1 cells. Thus, MMP-9 could be an appropriate therapeutic target to stop severe tissue damage caused by infection or chronic inflammation.