Cytosolic phospholipase A(2) (cPLA(2)) cleaves membrane phospholipids to release arachidonic acid, initiating lipoxygenase and cyclooxygenase pathways. Mice lacking a gene for cPLA(2) suggested important roles of the protein in allergic responses, fertility, and neural cell death. Here we show that cPLA(2) negatively regulates c-Myc expression in a B-Myb-dependent manner. Overexpression of cPLA(2) protein but not a mutant cPLA(2) protein that lacks in vitro binding ability with B-Myb inhibits B-Myb-dependent c-myc gene expression. The inhibition was associated with physical interaction of B-Myb protein with cPLA(2) both in the cytoplasm and the nucleus. Binding site analysis demonstrated that both the N and C termini of cPLA(2) interact with B-Myb. Macrophage colony stimulating factor (MCSF) stimulated cPLA(2) redistribution into the nucleus and also association with B-Myb in human monocytes. Importantly, macrophages from mice with a disrupted cPLA(2) gene demonstrated significantly increased levels of c-Myc protein in the nucleus compared with cells from the wild-type mice, whereas B-Myb levels were similar in the cells from the cPLA(2)(+/+) and cPLA(2)(-/-) mice. Moreover, an introduction of cPLA(2) into cPLA(2)(-/-) mouse macrophages resulted in decreased c-Myc protein levels, and an inhibition of cPLA(2) expression by small interfering RNAs or antisense RNA increased the c-myc transcription in macrophage colony stimulating factor-activated human monocytes. These findings provide new insights into the function of cPLA(2) in B-Myb-dependent gene expression.