AbstractKynurenine derivative 3‐hydroxyanthranilic acid (3‐HAA) is known to regulate the immune system and exhibit anti‐inflammatory activity by inhibiting T‐cell cytokine secretion and influencing macrophage activity. However, the definite role of 3‐HAA in the immunomodulation of hepatocellular carcinoma (HCC) is largely unexplored. An orthotopic HCC model and treated with 3‐HAA by intraperitoneal injection is developed. Furthermore, cytometry by time‐of‐flight (CyTOF) and single‐cell RNA sequencing (scRNA‐seq) analyses are carried out to define the immune landscape of HCC. It is found that 3‐HAA treatment can significantly suppress tumor growth in the HCC model and alter the level of various cytokines in plasma. CyTOF data shows that 3‐HAA significantly increases the percentage of F4/80hiCX3CR1loKi67loMHCIIhimacrophages and decreases the percentage of F4/80loCD64+PD‐L1lomacrophages. scRNA‐seq analyses demonstrate that 3‐HAA treatment is proved to regulate the function of M1 macrophages, M2 macrophages, and proliferating macrophages. Notably, 3‐HAA inhibits the proinflammatory factors TNF and IL‐6 in multiple cell subsets, including resident macrophages, proliferating macrophages, and pDCs. This study reveals the landscape of immune cell subsets in HCC in response to 3‐HAA, indicating that 3‐HAA may be a promising therapeutic target for HCC.