Objective: To investigate the effect of 4-hydroxysalicylaniline (RC17) on the proliferation and apoptosis of T lymphocyte leukemia cells, Jurkat and Hut-78, in vitro and in vivo. Methods: RC17 was used to treat Jurkat and Hut-78 cells with different concentration gradients. CCK8 assay and soft AGAR colony formation assay were used to detect the effect of RC17 on cell proliferation. The cell cycle and cell apoptosis were detected by flow cytometry. Immunofluorescence staining of γ-H2AX was used to detect the DNA damage. Proteins related to cell cycle, apoptosis and DNA damage were detected by Western blot. The mouse T-cell leukemia tumor model was constructed by subcutaneous injection of Jurkat cells, and RC17 was injected to observe the effect of drugs on tumor volume and body weight of mice. HE staining was used to observe the morphological changes of tumors and mouse organs. Immunohistochemical staining of Ki67 and γ-H2AX protein was used to analyze the effect of drugs on cell proliferation and DNA damage in tumors. Results: In Jurkat and Hut-78 cell lines, we demonstrated that RC17 could induce cell cycle arrest at S phase, induce cell apoptosis, and block DNA damage repair through phosphorylation of CHK1/CHK2 signaling pathway, both in vitro and in vivo. RC17 inhibited T cell leukemia tumors, and it had no obvious toxicity to mice. Conclusion: In conclusion, RC17 can be used as a potential anti-T-cell leukemia drug. The effect of RC17 on T-cell leukemia was accomplished through the CHK1/CHK2 signaling pathway.