Propofol, an intravenous general anaesthetic, exerts anaesthetic actions through interaction with γ-aminobutyric acid type A (GABAA) receptors in the supraspinal nervous system. However, whether propofol has any significant effects on synaptic transmission at the spinal level and whether it exhibits antinociceptive action is still not fully clarified. Spontaneous activity and stimulus-evoked responses of substantia gelatinosa (SG) neurones to noxious pinch stimuli were recorded using spontaneously breathing rats under propofol anaesthesia using in vivo whole-cell patch-clamp techniques. Precise actions of propofol on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) as well as excitatory postsynaptic currents (EPSCs) in SG neurones were also analyzed in spinal cord slice preparations. At clinical doses (5 mg/kg), propofol reversibly depressed action potentials elicited by noxious mechanical stimuli applied to the skin in the majority (6/8) of SG neurons recorded under in vivo conditions. This depression may have been caused by interactions of propofol with GABAA receptors, as decay time of GABAergic sIPSCs was prolonged after propofol injection (128 ± 11% of control, n = 5) with minimal effect on EPSCs. Although prolongation of IPSCs in vivo was reversible, IPSCs were progressively prolonged even after washout of propofol when the effect was tested using spinal cord slices. Propofol had a mild depressant effect on Aδ- and C-afferent-mediated EPSCs. We conclude that systemic bolus injection of propofol reversibly depressed nociceptive transmission, at least in part, by enhancing postsynaptic GABAA receptor-mediated responses in the SG.