ObjectiveTo provide a new insight into the diagnosis and treatment of hemiplegic shoulder pain (HSP) by investigating changes in serum pain mediators.DesignCross‐sectional study.Subjects/patientsShoulder pain group (n = 34) and control group (n = 21).MethodsPain‐free shoulder mobility, anxiety status, depression status, and shoulder pain were measured by passive range of motion (PROM), self‐rating anxiety scale, self‐rating depression scale (SDS), and visual analog scale, respectively. The enzyme‐linked immunosorbent assay was used to test the serum pain mediators, including interleukin (IL)‐1β, IL‐2, IL‐6, IL‐10, nerve growth factor (NGF), tumor necrosis factor‐α (TNF‐α), substance P (SP), calcitonin gene‐related peptide (CGRP), bradykinin (BK), 5‐hydroxytryptamine (5‐HT), prostaglandin E2 (PGE2), and lysophosphatidic acid (LPA).ResultsShoulder pain group pain‐free PROM significantly lower than control (p < .01), and SDS index score of shoulder pain group was significantly higher than control (p < .05). The rate of spasticity in the flexor elbow muscles is higher in shoulder pain group (p < .01). CGRP, IL‐10, and IL‐2 were significantly upregulated in shoulder pain group compared with control (p < .01), whereas NGF, TNF‐α, IL‐6, 5‐HT, PGE2, SP, LPA, BK, and IL‐1β were significantly decreased (p < .01).ConclusionPatients with HSP have a higher risk of joint mobility disorders and depression; spasticity may be an important factor in the development of shoulder pain; CGRP is thought to be the major pain mediator in HSP, and HSP may not be inflammatory.