Monocytes/macrophages play a prominent role in cutaneous wound healing. Persistent inflammation in diabetic wounds is associated with the inability of monocytic cells to switch from a phagocytic M1 (classically activated) to an anti-inflammatory, pro-regenerative M2 (alternatively activated) phenotype and as consequence, the proliferative phase of healing does not commence. A targeted cell therapy approach could potentially restore the pathological wound microenvironment through paracrine signalling to enable healing. This study investigated whether in vitro pre-treatment of monocytic (J774.1 A) cells - using a combination of endotoxin-induced immune tolerance (Pam3CSK4) and M2 polarization (IL-4) - could make these cells impervious to the pathological wound microenvironment and enhance the release of anti-inflammatory cytokines/growth factors. The effect of Pam3CSK4-induced tolerance and IL-4-associated polarization was assessed independently and in combination, on the expression of intracellular (flow cytometry) and secreted (ELISA) cytokines (TNF-ɑ, IL-6, IL-10, TGF-β) with and without re-stimulation to define the optimal pre-treatment conditions. Successive pre-treatment approach consisting of endotoxin tolerance followed by IL-4 priming, dampened TNF-ɑ release and induced intracellular TGF-β production upon re-stimulation. To mimic a chronic wound microenvironment, the J774A.1 monocytes were differentiated into macrophages using GM-CSF prior to pre-treatment (optimal condition) and subsequently exposed to diabetic wound fluid. The data demonstrated that in the presence of wound fluid, the successive pre-treatment, promoted M2 polarization (CD206) of monocytic cells and significantly dampened the intracellular production of both pro-inflammatory (TNF-ɑ, IL-6) and anti-inflammatory (IL-10, TGF-β) cytokines.