Propofol (2,6-diisopropylphenol) is a widely used intravenous anaesthetic with biological effects extending beyond transient loss of consciousness, including actions on mitochondrial function, immune responses, neuronal differentiation, axonal transport and synaptic plasticity. Every propofol-binding protein identified to date is membrane-embedded or intracellular, and whether the propofol scaffold engages extracellular signaling molecules is unknown. Using a hypnotically active biotinylated propofol analogue, we performed affinity enrichment from mouse brain followed by label-free LC–MS/MS, and identified Slit2, a secreted axon-guidance cue. Bio-propofol enriched endogenous Slit2 from brain lysate and Slit2–FLAG from conditioned medium, and free propofol competed this recovery. Docking analyses identified a favorable cavity on the concave face of the Robo-binding LRR2 domain. In Caenorhabditis elegans, slt-1(ok255) mutants were markedly resistant to propofol-induced paralysis. These convergent data identify Slit2 as a candidate extracellular propofol-associated protein and link Slit signaling to organismal propofol responsiveness.