The activity of α-type cytosolic phospholipase A (cPLA α, group IVA PLA ), which releases arachidonic acid (AA), is mainly regulated by the Ca -induced intracellular translocation/attachment of the enzyme to substrate membranes and its phosphorylation. We previously reported that tumor necrosis factor-α (TNFα) stimulated the formation of lactosylceramide (LacCer) in L929 fibroblast cells, and this lipid directly bound with and activated cPLA α [Nakamura et al. [2013] J. Biol. Chem. 288:23264-23272]. We herein investigated the role of phosphorylation signaling in the TNFα/LacCer-induced activation of cPLA α in cells. TNFα-treated L929 cells released AA via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cPLA α, while a treatment with LacCer alone released AA in a similar manner. The TNFα-induced responses including release of AA were decreased by the inhibition of LacCer synthesis. The treatment with TNFα and LacCer increased the levels of reactive oxygen species (ROS), and the reduction/scavenging of ROS decreased the phosphorylation cascade and release of AA in TNFα/LacCer-treated L929 cells. In the cell line CHO, the treatment with LacCer stimulated the phosphorylation cascade and release of AA via the formation of ROS. Treatments with the anti-LacCer antibody and 4β-phorbol 12-myristate 13-acetate stimulated the phosphorylation cascade, but did not release AA by itself. When combined with the Ca ionophore A23187, treatments with the anti-LacCer antibody and 4β-phorbol 12-myristate 13-acetate released AA. These results, including our previous findings, showed that LacCer alone simultaneously stimulates two processes to activate cPLA α: a phosphorylation signal and attachment of the enzyme to substrate membranes. J. Cell. Biochem. 118: 4370-4382, 2017. © 2017 Wiley Periodicals, Inc.