LPA (lysophosphatidic acid) is a potent bioactive phospholipid, which regulates a number of diverse cellular responses through G-protein coupled LPA receptors. Intracellular LPA is generated by the phosphorylation of monoacylglycerol by acylglycerol kinase (AGK); however the role of intracellular LPA in signaling and cellular responses remains to be elucidated. Here, we investigated signaling pathways of IL-8 secretion mediated by AGK and intracellular LPA in human bronchial epithelial cells (HBEpCs). Expression of AGK in HBEpCs was detected by real-time PCR and over-expressed AGK was mainly localized in mitochondria as determined by immunofluorescence and confocal microscopy. Over-expression of lentiviral AGK wild type increased intracellular LPA production (~1.8 fold), enhanced LPA-mediated IL-8 secretion, and stimulated tyrosine phosphorylation EGF-R. Further, down-regulation of native AGK by AGK siRNA decreased intracellular LPA levels (~2 fold) and attenuated LPA-induced p38MAPK, JNK and NF-B activation, tyrosine phosphorylation of EGF-R, and IL-8 secretion. These results suggest that native AGK regulates LPA-mediated IL-8 secretion involving MAPKs, NF-B and transactivation of EGF-R. Thus AGK may play an important role in innate immunity and airway remodeling during inflammation.