Abstract: Metabolic abnormalities in mammalian joint tissues are closely linked to various diseases, including rheumatoid arthritis and osteoarthritis. The variations of small molecule metabolites within bone joint tissues can affect cell proliferation, drive the secretion of inflammatory mediators, mediate leukocyte infiltration, and consequently induce synovial inflammation and cartilage damage. Therefore, the development of novel mass spectrometry imaging (MSI) methods for visualizing metabolites in joint tissues is crucial for comprehensively understanding the metabolic characteristics of different microregions under both physiological and pathological conditions. In this study, an airflow-assisted desorption electrospray ionization (AFADESI)-MSI was used to establish an ambient MSI method for rat joint tissues. To ensure the effectiveness of the method, dynamic range, sensitivity, and imaging effect were selected as the primary evaluation criteria. The tissue preparation, section thickness, and spray solvent systems were systematically optimized. Compared to the AB glue transfer method, the tape adhesion method can obtain complete bone joint tissue sections and clearer images. A section thickness of 20 μm, as opposed to 15 μm, provides higher ion intensity in the m/z 600-900 range. Among the tested spray solvent systems, compared with methanol-water (8:2, V/V), acetonitrile-water (8:2, V/V), and the acetonitrile-isopropanol-water (6:2:2, V/V/V), the acetonitrile-isopropanol-water (4:4:2, V/V/V) prove to be the most effective, detecting the highest number of metabolites and lipids when employed as the spray solvent. The precision of this method was evaluated on six adjacent rat knee sections under positive and negative ion modes, with the relative standard deviation (RSD) for the ion intensity of representative metabolites being less than 20%. Subsequently, this method was utilized for spatially resolved metabolomics analysis of rat knee tissues. A total of 613 metabolites are annotated in the overall knee tissues. Based on the optical images, the rat knee tissues were segmented into three microregions for bone, marrow, and cartilage. 452, 418, and 451 metabolites in these microregions are identified, respectively. In bone, the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and the TCA cycle are more prominent. In bone marrow, ascorbate and purine metabolism pathways are more significant. In cartilage, arginine and proline metabolism, as well as lysine degradation, are more pronounced. These findings highlight significant metabolic heterogeneity across different microregions of rat knee joint tissues. Overall, this study develops an AFADESI-MSI method for in-depth profiling of metabolites within rat joint tissues, providing a powerful tool for in situ visualization of metabolites in metabolic studies of joint tissues.