The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40-50 μg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP's ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.