Diets supplemented with walnuts have shown to protect brain against oxidative and inflammatory cytotoxicity and promote protective cellular and cognitive function. The current study was undertaken to test the hypothesize that whole walnut extract inhibits LPS-induced microglial activation by regulating calmodulin (CaM) expression through [Ca2+]i. To test this hypothesis, we used an in vitro model the highly aggressively proliferating immortalized (HAPI) cells, a rat microglial cell-line, treated with various concentrations of walnut extracts (WNE). Treatment with walnut extract (1.5, 3 or 6%) induced a slow rise in intracellular calcium in a concentration- and time-dependent manner, and this rise became exaggerated when cells were depolarized with potassium chloride (KCl, 100 mM). Cells treated with walnut extract (1, 3 or 6%) up-regulated calmodulin (CaM) protein levels, with one hour post-treatment being the peak time, regardless of walnut extract concentration. Interestingly, this walnut extract-induced up-regulation of CaM was blocked by pre-treatment with thapsigargin. Additionally, treatment with walnut extract (1, 3 or 6%) one hour prior to lipopolysaccharide (LPS) treatment was found to be effective in preventing LPS-induced up-regulation of inducible nitric oxide synthase (iNOS) expression, up-regulation of ionized Ca2+-binding adaptor-1 (IBA-1), and down-regulation of CaM. These findings suggest that bioactive compounds in walnut are capable of modulating microglial activation through regulation of intracellular calcium and CaM expression. Nutritional interventions using walnuts may be effective in the amelioration of chronic inflammation and neurodegeneration.