In a recent mass spectrometry screen we have identified 108 new proteins that were modified endogenously by covalent binding of biotin; members of the heat shock superfamily of proteins, including heat shock protein 72 (HSP72), were overrepresented among the biotinylated proteins. Mammals respond to infections by secreting extracellular HSP72 (eHSP72) which elicits an immune response. Here, we identified five biotinylation sites in HSP72 using mass spectrometry and site-directed mutagenesis. We used co-immunoprecipitation, mass spectrometry, and limited proteolysis assays to demonstrate that HSP72 interacts physically with the protein biotin ligase, holocarboxylase synthetase (HLCS) leading to the biotinylation of residues K112, K128 K348, K361, K415, and probably additional lysines. Finally, we demonstrated that HLCS-dependent biotinylation of eHSP72 increases the expression of the chemokine RANTES by the HEK293 human embryonic kidney cells. In conclusion, we report a novel endogenous modification of HSP72 and demonstrated that the binding of biotin to eHSP72 poises cells for a strong immune response.