Mucosa-associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly activate the nuclear factor (NF)-kappaB pathway. Gastric MALT lymphomas harboring such translocations usually do not respond to Helicobacter pylori eradication, while most of those without translocation can be cured by antibiotics. To understand the molecular mechanism of these different MALT lymphoma subgroups, we performed gene expression profiling analysis of 21 MALT lymphomas (13 translocation-positive, 8 translocation-negative). Gene set enrichment analysis (GSEA) of the NF-kappaB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions have shown that translocation-positive MALT lymphomas are characterized by an enhanced expression of NF-kappaB target genes, particularly toll like receptor (TLR)6, chemokine, CC motif, receptor (CCR)2, cluster of differentiation (CD)69 and B-cell CLL/lymphoma (BCL)2, while translocation-negative cases were featured by active inflammatory and immune responses, such as interleukin-8, CD86, CD28 and inducible T-cell costimulator (ICOS). Separate analyses of the genes differentially expressed between translocation-positive and -negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. Finally, expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10-mediated NF-kappaB activation in vitro. Our findings provide novel insights into the molecular mechanism of MALT lymphomas with and without translocation, potentially explaining their different clinical behaviors.