Objectives: Knee osteoarthritis is a leading global cause of morbidity. This study investigates the effects of knee synovial fluid from patients with osteoarthritis (OA-SF) on the activity of dorsal root ganglion sensory neurons that innervate the knee (knee neurons) as a novel translational model of disease mediated nociception in human osteoarthritis. Methods Dissociated cultures of mouse knee neurons were incubated overnight or acutely stimulated with OA-SF (n=4) and fluid from healthy donors (n=3, Ctrl-SF). Electrophysiology and Ca2+-imaging determined changes in electrical excitability and transient receptor potential (TRP) channel function respectively. Results Incubation with OA-SF induced knee neuron hyperexcitability compared to Ctrl- SF: the resting membrane potential significantly increased (F(2,92) = 5.6, p = 0.005, ANOVA) and the action potential threshold decreased (F(2,92) = 8.8, p = 0.0003, ANOVA); TRPV1 ( F(2,445) = 3.7, p = 0.02) and TRPM8 (F(2,174) = 11.1, p < 0.0001, ANOVA) channel activity also increased. Acute application of Ctrl-SF and OA-SF increased intracellular Ca2+ concentration via intra- and extracellular Ca2+ sources. Conclusions Human OA-SF acutely activated knee neurons and induced hyperexcitability indicating that mediators present in OA-SF stimulate sensory nerve activity and thereby give rise to knee pain. Taken together, this study provides proof-of-concept for a new method to study the ability of mediators present in joints of patients with arthritis to stimulate nociceptor activity and hence identify clinically relevant drug targets for treating knee pain.