The purpose of this study is to investigate whether a near-infrared fluorescence (NIRF) probe, Cy5.5-d-glucosamine (Cy5.5-2DG), can image arthritis in collagen-induced arthritic (CIA) mice. The presence of arthritis was verified by both visual examination and micro-computed tomography (MicroCT) imaging. CIA mice were imaged by a micro-positron emission tomography (MicroPET) scanner one hour after intravenous injection of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG). After radioactivity of [¹⁸F]FDG decayed away, Cy5.5-2DG was injected into a lateral tail vein of the mice. Arthritic tissue targeting and retention of Cy5.5-2DG in CIA mice were evaluated and quantified by an optical imaging system. Inflammatory tissue in CIA mice was clearly visualized by [¹⁸F]FDG-MicroPET scan. NIRF imaging of Cy5.5-2DG in the same mice revealed that the pattern of localization of Cy5.5-2DG in the arthritic tissue was very similar to that of [¹⁸F]FDG. Quantification analysis further showed that [¹⁸F]FDG uptake in arthritic tissues at one hour post-injection (p.i.) and Cy5.5-2DG uptakes at different time points p.i. were all well correlated (r² over 0.65). In conclusion, Cy5.5-DG can detect arthritic tissues in living mice. The good correlation between the [¹⁸F]FDG uptake and Cy5.5-2DG accumulation in the same arthritic tissue warrants further investigation of Cy5.5-2DG as an approach for assessment of anti-inflammatory treatments.