Upon stimulation by distinct bacterial/viral products/agonists, APCs including macrophages tend to express particular TLR molecules to coordinate the signaling that ultimately target at chromatin and mediate the activity of downstream transcriptional factors in regulating characteristic sets of gene expression for innate immune response. To investigate largely unknown regulatory mechanism underlying agonist-specific TLR-mediated innate immune responses, at subcellular resolution, we first analyzed Pam3CSK4-induced proteome changes in living macrophages and identified the differentially expressed proteins in the cytosol and chromatin-associated fractions, respectively, by using AACT/SILAC-based quantitative proteomic approach. In the cytosol fraction, we found that the proteins with notable Pam3CSK4-induced expression changes were primarily involved in post-translational events, energy metabolism, protein transporting, and apoptosis. Among them, a ubiquitous and highly conserved iron-binding protein, Ferritin, was further characterized as a modulator for the expression of a TLR2-specific cytokine IL-10 in murine macrophage cells by using small-interfering RNA (siRNA). Interestingly, we simultaneously identified multiple apoptosis-related proteins showing opposite trend in their regulated expressions, which clearly indicated the existence of systems regulation in differentially modulating the signal for the cross-road balance between protecting cell from apoptosis and the apoptosis of infected cells. For those regulated proteins identified in the nuclear fraction, we integrated bioinformatics to find the interactions of certain chromatin-associated proteins, which suggested their interconnected involvements in proteasome-ubiquitin pathway, DNA replication, and post-translational activity upon Pam3CSK4 stimulation. Certain regulated proteins in our quantitative proteomic data set showed the similar trend of up-regulation in both Pam3CSK4- and LPS-stimulated macrophages (Nature 2007, 447, 972), suggesting their belonging to the recently identified class of pro-inflammatory genes. The regulatory discrepancy between both data sets for other set of genes indicated their agonist-specific nature in innate immune responses.