Functional proteomics is a useful method to explore changes in protein expression in human diseases, including carcinomas. To identify tumor-associated proteins as biomarkers or molecular targets of human oral squamous cell carcinomas (OSCCs), we performed two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Comparison of the protein expression profiles of OSCC cell lines and normal oral keratinocytes identified six proteins with markedly different expression levels. Of the six proteins, we found a 1D-myo-inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) protein that was down-regulated in OSCC cell lines. ITPKA phosphorylates inositol 1,4,5-trisphosphate, which regulates the calcium (Ca2+) level within the cell by releasing Ca2+ from intracellular stores, and is responsible for regulating the levels of a large number of inositol polyphosphates that are important in cellular signaling. Western blots revealed dramatically down-regulated ITPKA expression in all OSCC cell lines examined. Real-time quantitative reverse transcriptase-polymerase chain reaction showed down-regulated ITPKA mRNA expression in nine of 12 (75%) OSCC cell lines. Immunohistochemistry analysis showed that 40 of 100 OSCC clinical samples had a significant decrease in ITPKA. Poorly differentiated tumors showed significantly lower immunoreactivity of the protein compared to well- and moderately-differentiated tumors. These data suggest that ITPKA may be related to carcinogenesis by the modulation of inositol polyphosphates and Ca2+ homeostasis and that ITPKA may be a potential novel molecular target, biomarker, parameter, or all of these of cellular differentiation and of intracellular Ca2+ homeostatic characteristics in clinical medicine.