Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in chronic inflammatory disease, but its role in spinal diseases is unknown. The purpose of this study is to investigate the role of CX3CL1/CX3CR1 chemokine on hypertrophy of the ligamentum flavum (LF) in degenerative lumbar stenosis (DLS) compared with that of non-degenerative spinal condition (NDS) of the lumbar spine and correlation between expression of CX3CL1/CX3CR1 chemokine and thickness of LF. The mRNA concentrations of CX3CL1/CX3CR1 chemokine were analyzed in the surgically obtained LF specimens from DLS (n = 10) and NDS (n = 11) by real-time PCR. The localization of CX3CL1/CX3CR1 chemokine within the LF was determined using immunohistochemical study. Plasma levels of soluble FKN (sFKN) were measured by enzyme-linked immunosorbent assay, respectively. The thickness of the LF was measured with axial T1-weighted MRI. The cells that express CX3CL1/CX3CR1 chemokine ratio in the LF observed in DLS group were substantially higher than in NDS group. In ELISA, the plasma levels of sFKN was significantly increased in DLS compared with patients in the other groups (p = 0.006). There was greater CX3CL1/CX3CR1 expression in DLS as quantified by RT-PCR (p = 0.004, 0.010). Thickness of LF in patients was significantly correlated with serum CX3CL1 level (R(2) = 0.824, p = 0.003) and with mRNA expression of CX3CL1/CX3CR1 (R(2) = 0.671, p = 0.000) (R(2) = 0.514, p = 0.001). This study identified for the first time that increases in CX3CL1 and CX3CR1-expressing cells are significantly related to LF hypertrophy.