The invasion-associated type III secretion system of Salmonella enterica assembles as a supra-molecular structure, termed needle complex, which spans the bacterial envelope. Here, we present evidence for protein-peptidoglycan interactions that modulate the assembly of this organelle. The presence of major membrane components of the needle complex (PrgH, PrgK and InvG) and InvH, required for efficient assembly of the organelle, was examined in peptidoglycan purified by extensive boiling of bacteria in 4% SDS. InvH, PrgH and PrgK, but not InvG, were detected in this purified material. InvH was present in the peptidoglycan in higher relative amounts than PrgH or PrgK, and was the only protein efficiently bound to peptidoglycan in cross-linking experiments. Analysis in mutants defective for needle complex proteins showed that the needle proteins PrgI and PrgJ and, to a lesser extent, InvH, sustain the association of PrgH and PrgK with peptidoglycan. In contrast, the association of InvH with peptidoglycan did not necessitate other needle complex proteins. Functional analysis showed that the association of InvH, PrgH and PrgK with peptidoglycan is abolished in live bacteria carrying structural modifications in the peptidoglycan. The loss of these interactions caused a marked reduction in the number of needle complexes and, concomitantly, in protein secretion and bacterial invasion of cultured eukaryotic cells. Altogether, these data provide the first evidence for an association between proteins of the Salmonella needle complex and the peptidoglycan. In addition, we demonstrate that these protein-peptidoglycan interactions are critical for an efficient and correct assembly of this specialized organelle.