Introduction: Mesenchymal stromal cells (MSCs) reduce inflammation and improve recovery after myocardial infarction in mice and are thus being increasingly investigated for their immunomodulatory capabilities. Importantly, MSC spheroids highly express the anti-inflammatory proteins TSG-6 and COX-2 which polarise pro-inflammatory macrophages to an anti-inflammatory phenotype. Preliminary microarray data from our laboratory have shown that MSC spheroids also upregulate the expression of matrix metalloproteinases (MMPs), enzymes that degrade matrix proteins but may also have a role in regulating inflammation. The aim of this study was to determine how MMPs regulate the expression of TSG-6 and COX-2 and thus the anti-inflammatory properties of MSC spheroids. Methods: MSCs were cultured as spheroids in the presence or absence of siRNA targeted to specific MMPs. Expression levels of MMPs and inflammatory cytokines were assessed using qRT-PCR and western blot. Conditioned media from knockdown spheroids were tested for their ability to induce anti-inflammatory macrophage polarisation. Results: Knockdown of MMP-1 or MMP-8 significantly decreased anti-inflammatory TSG-6 and COX-2 protein expression whereas knockdown of MMP-9 significantly increased anti-inflammatory TSG-6 expression in MSC spheroids. Conditioned media from MMP-1 knockdown spheroids, which lack TSG-6 and COX-2, were unable to induce anti-inflammatory macrophage polarisation. In vivo work also showed that intra-peritoneal injection of MSC spheroids reduced the inflammatory reaction of mice to the pro-inflammatory agent zymosan. Conclusion: MMPs have an important and previously unknown role in regulating the anti-inflammatory properties of MSC spheroids. A deeper understanding of specific MMPs mechanism of action could lead to potential MSC-based anti-inflammatory therapeutics for myocardial infarction.