There are two types of vascular smooth muscle contraction. One is normal contraction, which is physiological and Ca2+-dependent. Another is abnormal contraction, which causes vasospasm and Ca2+-independent. Rho-kinase and PKC are known key molecules to mediate the signal transduction of abnormal vascular smooth muscle contraction. Sphingosylphosphorylcholine (SPC) is a member of sphingolipids and induces Ca2+-independent, Rho-kinase-mediated abnormal vascular smooth muscle contraction via the activation of Src family tyrosine kinase (Src-TK). We found SPC-induced contraction is cholesterol-dependent and suggest the involvement of membrane raft in the signal transduction of abnormal vascular smooth muscle contraction mediated by SPC/Src-TK/Rho-kinase pathway. Eicosapentaenoic acid specifically inhibited abnormal vascular smooth muscle contraction through the inhibition of SPC/Src-TK/Rho-kinase pathway. By functional proteomics, we identified cytoskeleton-related proteins as the candidate of novel molecule to mediate abnormal vascular smooth muscle contraction and investigating their interaction with Rho-kinase. We hope that this interaction could be a new drug target.