Diffuse noxious inhibitory control (DNIC) works through the “pain-inhibits-pain” principle in which an additional painful (conditioned) stimulus can suppress the initial experienced pain through the descending and inhibiting pathways. Painful stimulation produced less pain inhibition in patients with knee osteoarthritis patients (KOA) than in controls, suggesting an impaired DNIC function and a loss of endogenous pain modulation. Electroacupuncture (EA) is widely used to treat acute pain associated with KOA, but the available evidence of its benefit on chronification of acute pain is scarce. This is a single-arm clinical study aims to evaluate the effect of EA on the chronification of pain associated with KOA and provide a profile of various cytokines underlying the pathogenesis of KOA. Participants are recruited through hospital-based recruitment and advertisements, diagnosis was based upon the criteria formulated by the American College of Rheumatology. Each participant was administered with EA (2 mA < current < 5 mA) at the ipsilateral EX-LE5, ST35, ST34 and SP10 for two weeks (once a day, 30 minutes per session, in 5 sessions per week). Visual Analog Scale (VAS), DNIC function, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Numerical Rating Scale (NRS), Emotional Scale (ES) and Present Pain Intensity (PPI) are evaluated before treatment and after 5 to 10 sessions of treatment. Cytokines including GRO, TNF-α, VEGF, IP-10, IL-1β, IL-17, IL-8, MCP-1 and IL-10 levels in plasma were measured using a Human Cytokine/Chemokine Magnetic Bead Panel on MAGPIX instrument before and after two weeks of treatment. A total of 39 patients with KOA were enrolled in our study (age: 63.46±9.89 years; height: 1.63±0.07 m; BMI: 22.83±2.89), all of them completed the trial. After 5 sessions of EA treatment, a significant decrease of VAS, WOMAC scores, NRS, ES and PPI was detected, but no significant difference in DNIC was observed. After two weeks' treatment, all clinical parameters (VAS, DNIC, WOMAC, NRS, ES, PPI) reduced significantly when compared with baseline; GRO, IL-17A, IL-1b, IL-8, MCP-1, TNF-a, VEGF levels in plasma reduced significantly while IL-10 and IP-10 concentrations were elevated. This study appeared to provide evidence that EA was effective in improving chronic pain associated with KOA through repairing the impaired DNIC function and down-regulation of OA detrimental cytokines. A randomized controlled prospective study with large sample size is required to clarify the effect of EA in reversing the chronification of pain in KOA.