Sphingosine-1-phosphate (S1P) is an important bioactive lipid mediator in inflammation. There is evidence that S1P increases the excitability of nociceptors, however, its role in spontaneous inflammatory pain is still unknown. Here, we examine spontaneous pain-like behaviour in mice in vivo after local injectionof S1P and the excitatory effect of S1P on nociceptive neurons in vitro. We performed behavioural studies to measure the number of flicks and licking duration in control S1P₁ receptor flox/flox (S1P₁^fl/fl) and conditional transgenic mice which selectively lack the S1P₁ receptor subtype in Na_v1.8 expressing nociceptive neurons (SNS-S1P₁^-/-). The direct effect of S1P on sensory neurons was investigated by single fibre and patch clamp recordings and microfluorimetric Ca²⁺ measurements. S1P increased the flicking and licking behaviour both in S1P₁^fl/fl and SNS-S1P₁^-/- mice. Furthermore, S1P induced small inward currents, Ca²⁺ transients and AP firing in cultured neurons. In the absence of extracellular Ca²⁺ the S1P-mediated Ca²⁺ transients were not detectable and they were also blocked by SKF96365, an inhibitor of non-capacitive Ca²⁺ entry. In presence of the G-protein inhibitor GDP-b-S the S1P-evoked current was completely abolished suggesting a G-protein dependent mechanism. Together, our data support the idea that the S1P evokes spontaneous pain-like behavioural via G-protein dependent activation of an excitatory inward current, which however, does not involve activation of the S1P₁ receptor expressed in nociceptive neurons.