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Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy. Author summary: The gram-negative bacterium Neisseria meningitidis can cause live-threatening meningitis in humans after crossing the blood-brain barrier (BBB). During infection, N. meningitidis may invade brain endothelial cells as a first step to cross the BBB. The bioactive lipid Sphingosine 1-phosphate (S1P) is an important regulator of BBB integrity during health and disease. While interaction of S1P with S1P receptor (S1PR) 1 enhances barrier stability, activation of S1PR2 leads to barrier leakage. We found that N. meningitidis induces S1P release from brain endothelial cells in parallel with increased expression of S1PR2 and downstream activation of the epidermal growth factor receptor, indicating a shift in the S1PR signalling balance during infection. Moreover, we showed that inhibition of S1P production or S1PR2 signalling reduced bacterial uptake and pro-inflammatory cytokine release. Conversely, pharmacological activation of S1PR1 and 3 had an even higher protective effect against bacterial invasion, indicating that the S1P/S1PR axis is actively manipulated by N. meningitidis and thus highlighting potential drug targets to prevent meningococcal meningitis. [ABSTRACT FROM AUTHOR]