Tuberculous and non-tuberculous mycobacteria release extracellular vesicles (EVs) containing proteins, lipids and nucleic acids. The Mycobacterium marinum-zebrafish model is a widely studied model for human tuberculosis as it mimics granuloma formation. EVs from M. marinum cultures were isolated and purified by density gradient centrifugation. Naïve THP-1 cells were treated with a high dose (HD-10µg protein equivalent of purified M. marinum EVs) and a low dose (LD-1µg) or PBS control for 48 and 72 hours and changes in gene expression were measured by Affymetrix microarray analysis. Comparison of gene expression across all conditions (False Discovery Rate F-test p value <5.00E-09 as significant) demonstrated significant changes for 84 genes mainly involved in inflammatory responses such as chemokine and cytokine signalling, toll receptor signalling and cellular growth pathways including amino acid biosynthesis, apoptosis signalling and p38 MAPK pathways. Pairwise comparisons between HD, LD and untreated PBS controls at both time points identified 5,388 gene with significant changes in at least one of the analyses (p value <0.05 and ≥ 2-fold change). Genes with drastic fold-changes include CCL2, C3, TNFAIP3, CYBB, ALOX5AP, and are involved in inflammatory responses against invading M. tuberculosis. THP1-Dual macrophages (obtained by phorbol 12-myristate 13-acetate (PMA) conversion) were treated with HD and LD M. marinum EVs and were found to significantly activate the NF-κB pathway (as indicated by an increase in secreted embryonic alkaline phosphatase (SEAP) expression) but not found to activate IRF pathway (as indicated by no change in Lucia Luciferase (LL) expression). Elevation of secreted CXCL10/IP10 was not observed in the treated cells. In conclusion, M. marinum EVs were found to alter host gene expression and stimulate the NF-κB pathway which gives an indication to the potential of EVs as targets for new vaccines and drug development for the management of tuberculosis.