The cellular mechanisms that directly regulate the production of pro- and anti-inflammatory cytokines after lipopolysaccharide (LPS) stimulation in mast cells are currently unresolved. The aim of this study was to clarify the role of phosphatidylinositol 3-kinase (PI3K) in the production of IL-12 and IL-10 in mouse bone marrow-derived mast cells (BMMCs), stimulated with Escherichia coli-derived LPS. LPS activates the PI3K signalling pathway; analysis of cytokine production following LPS stimulation of BMMCs revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 syntheses. IL-12 production was enhanced, whereas IL-10 levels were suppressed. Inhibition of LPS-mediated activation of the PI3K pathway resulted in a pronounced reduction of NF-κB activity that was dependent on IκBα phosphorylation. These findings demonstrate a regulatory function for PI3K in modulating IL-10 and IL-12 production in mast cells and provide insight into how engagement of the PI3K pathway affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of early inflammation.