Glucocorticoids, or corticosteroids, are effective treatments for many chronic inflammatory diseases and, in mild/moderate asthma, long-acting β₂-adrenoceptor agonists (LABAs) enhance the efficacy of inhaled corticosteroids (ICSs) more than increasing the ICS dose. In human bronchial epithelial, BEAS-2B, cells, expression of TNFα-induced protein 3 (TNFAIP3), or A20, a dual-ubiquitin ligase that provides feedback inhibition of NF-B, was induced by budesonide, an ICS, formoterol, a LABA, and was further enhanced by budesonide/formoterol combination. The pro-inflammatory cytokine, TNF, induced TNFAIP3 and TNF expression. Whereas subsequent budesonide treatment enhanced TNF-induced TNFAIP3 and reduced TNF expression, formoterol amplified these differential effects. In primary human airway smooth muscle cells, TNFAIP3 expression was induced by TNF. This was largely unaffected by budesonide, but was acutely enhanced by budesonide/formoterol combination. In BEAS-2B cells, TNF recruited RELA, the main NF-B transactivating subunit, to a 3' region of the TNF gene. RELA binding was reduced by budesonide, further reduced by formoterol co-treatment, and was associated with reduced RNA polymerase II recruitment to the TNF gene. This is consistent with reduced TNF expression. TNFAIP3 knockdown enhanced TNF expression in the presence of TNF, TNF plus budesonide and TNF plus budesonide/formoterol combination and confirms feedback inhibition. A luciferase reporter containing the TNF 3' RELA binding region recapitulated TNF inducibility and was inhibited by an IB kinase inhibitor and TNFAIP3 over-expression. Repression of reporter activity by budesonide was increased by formoterol and involved TNFAIP3. Thus LABAs may improve the anti-inflammatory properties of ICSs by augmenting TNFAIP3 expression to negatively regulate NF-B.