γ-Tilmanocept ( Tc-tilmanocept) is a receptor-directed, radiolabeled tracer that is FDA-approved for guiding sentinel lymph node biopsy. Tilmanocept binds the C-type lectin mannose receptor (MR, CD206) on macrophages. In this study, nonradioactive, fluorescently-labeled Cy3-tilmanocept was used to detect CD206 mononuclear cells in the cartilage of mice with antibody-induced arthritis and in the synovial fluid and tissue of human subjects with rheumatoid arthritis (RA) for comparison with osteoarthritis (OA), and healthy volunteer (HV) controls. Murine arthritis was induced by injection of monoclonal anti-cartilage antibody followed by injection of Escherichia coli lipopolysaccharide. Post-arthritis development (7-11 days), the mice were injected intravenously with Cy3-tilmanocept followed by in vivo and ex vivo epifluorescence imaging. Two-photon imaging, immunofluorescence, and immunohistochemistry were used to identify articular and synovial macrophages (CD206, F4/80, and Cy3-tilmanocept binding) in murine tissues. Cy3-tilmanocept epifluorescence was present in arthritic knees and elbows of murine tissues; no radiographic changes were noted in the skeletons. However, inflammatory arthritic changes were apparent by histopathology and immunohistochemistry (F4/80), immunofluorescence (CD206) and Cy3-tilmanocept binding. In human RA synovial fluid, Cy3-tilmanocept staining correlated with CD206 /CD16 cells; negligible labeling was observed in OA samples. Cy3-tilmanocept colocalized with CD206 and staining was significantly higher in RA synovial tissue compared to OA or HV. Our results demonstrate that imaging with Cy3-tilmanocept can detect in vivo inflammatory, CD206 macrophages in an early arthritis animal model and in human RA patients. These data establish a novel tool for preclinical research of early arthritis and have implications for early RA detection and monitoring of therapeutic efficacy in humans.