Misgurnus anguillicaudatus, also known as oriental weather loach, is widely consumed and favored in East Asia due to its superior nutritional values and excellent flavor. In this study, a crude Misgurnus anguillicaudatus carbohydrates (MAC) was isolated from Misgurnus anguillicaudatus. Subsequently, two parts, which were named MAO and MAP, respectively, were separated from MAC, and their primary structures and immunomodulatory activity were investigated. The results showed that MAO had a molecular weight of 2854 Da, and principally consisted of arabinose (77.11%) and rhamnose (21.97%), together with minor levels of fucose (0.92%); MAP, with a molecular weight of 3873 Da, was mainly composed of fucose (87.55%) and a small amount of rhamnose (8.86%) and galactose (3.59%). The in vitro assay showed that MAC could significantly enhance the proliferation of macrophages without cytotoxicity and increase the production of immune substances (TNF-α, IL-6). Together with Western blot results, we speculated that MAC could stimulate RAW264.7 murine macrophage cells to secrete TNF-α and IL-6 through up-regulating TLR4-MAPK-p38 signaling pathways. The results indicated that MAC could be a potential immune agent and might provide meaningful information for further chain conformation and immune mechanism research.