The role of Salmonella typhimurium type III secretion system (T3SS-1)-translocated proteins in chemokines' expression and protein phosphorylation was investigated in HeLa cells. Infection of HeLa cells with S. typhimurium activated IL-8 and GRO-alpha expression at higher levels than infection with a S. typhimurium sipAsopABDE2 mutant, confirming that T3SS-1-secreted proteins are required to fully induce chemokine expression in HeLa cells. A S. typhimurium sipAsopABDE2 mutant complemented with sipA or a strain carrying a chromosomal copy of sipA (sopABDE2 mutant) activated chemokines at significantly higher levels than a S. typhimurium sipAsopABDE2 mutant. However, extracellular addition of recombinant SipA failed to induce IL-8 expression. Phosphorylation analyses revealed that S. typhimurium induced a twofold increase in the phosphorylation of B23, CREB1, ERK1, JUN, p38MAPK, and NR1. JUN and p38MAPK were phosphorylated by S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant) while none was more than twofold phosphorylated in cells infected with the S. typhimurium sipAsopABDE2 mutant. Treating cells with JUN and p38MAPK inhibitors significantly decreased IL-8 expression in sopABDE2 mutant infected cells. These data indicate that S. typhimurium SipA induces expression of CXC chemokines through phosphorylation of IL-8-transcription regulatory proteins, JUN and p38MAK.