4-Hydroxycanthin-6-one is a novel quinoline alkaloid isolated from the stem bark of Ailanthus altissima. Additionally, five other known compounds were identified. The structure of the new compound was established through analysis of physical and spectral data, and its absolute configuration was determined using electronic circular dichroism and quantum chemical calculations. These compounds exhibit significant inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells, indicating potential anti-inflammatory properties.