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Objective: To exploit the elevated glutathione (GSH) levels in the tumor microenvironment and investigate the therapeutic efficacy of a novel glutathione-responsive apurinic/apyrimidinic (AP) site captor, Probe-NEt, against anaplastic thyroid cancer (ATC). Methods: Fluorescence imaging compared Probe-NEt uptake and activation in normal thyroid (Nthy ori 3-1), ATC (THJ-16T, CAL-62), and lung cancer (H1299) cells. Half maximal inhibitory concentration (IC₅₀) values were determined by cytotoxicity assays; DNA damage was evaluated using appropriate assays. Flow cytometry analyzed cell cycle distribution and apoptosis following treatment with low (5 μmol/L) or high (20 μmol/L) Probe-NEt concentrations. BALB/c nude mice bearing subcutaneous ATC xenografts received low (0.025 mg) or high (0.05 mg) dose injections. Tumor volumes were monitored; HE staining assessed biosafety in major organs; immunohistochemistry detected apoptosis-related protein expression. Results: ATC cells demonstrated significantly higher Probe-NEt activation than normal thyroid cells. Probe-NEt exhibited selective cytotoxicity (higher IC₅₀ in normal vs. ATC cells; all P＜0.01) with time-dependent characteristics; the selectivity ratio increased from 1.7 at 24 h (62.4 vs. 37.7 μmol/L) to 2.4 at 48 h (32.7 vs. 13.5 μmol/L). Probe-NEt induced DNA damage, G₂/M arrest (THJ-16T: from 5% to 43%; CAL-62: from 19% to 37%), and dose-dependent late apoptosis. In THJ-16T cells, late apoptotic rates rose from 5.49% (control) to 13.95% (low-dose) and 63.43% (high-dose), with viable cells decreasing accordingly (89.42%, 76.01%, 20.45%). CAL-62 cells showed similar trends (16.72%, 40.19%, 69.88%). In vivo, Probe-NEt significantly suppressed tumor growth without hepatorenal toxicity (all P＞0.167). Immunohistochemistry revealed upregulated pro-apoptotic proteins, downregulated anti-apoptotic proteins, and decreased Ki-67 expression. Conclusion: The glutathione-responsive AP site captor Probe-NEt significantly inhibits ATC cell growth, induces G₂/M phase cell cycle arrest, promotes late apoptosis, and exhibits high selectivity and favorable biosafety profiles.