<p>Tetrodotoxin (TTX) is a potent neurotoxin with therapeutic potential, particularly in the fields of analgesia, cancer pain treatment, and drug addiction therapy. However, its high toxicity and lack of antidotes limit its clinical application. This study evaluated the acute toxicity and sub-acute toxicity of TTX via intramuscular injection in Sprague–Dawley (SD) rats. The acute toxicity test employed the Bliss method to determine the median lethal dose (LD₅₀), with 10 rats per group (sex-balanced). Rats received a single intramuscular injection at doses of 8.2–20.0&#xa0;μg/kg. The sub-acute toxicity study was conducted through daily intramuscular injections at doses of 1.5, 3.0, and 6.0&#xa0;μg/kg/day for 28 consecutive days. Parameters, including body weight, food consumption, hematology, serum biochemistry, urinalysis, and histopathology, were assessed. Acute toxicity was characterized by squinting, reduced spontaneous activity, convulsions, hind limb rigidity, limb weakness, and mortality. In the sub-acute toxicity study, no toxicity-related changes associated with TTX treatment were observed in any dose group compared to controls, including in food consumption, hematology, urinalysis, and histopathological examination. Although statistically significant differences in body weight, serum biochemical, and organ weights were noted at certain time points in individual dose groups, these were not dose-dependent and were of minimal magnitude. The LD₅₀ of TTX administered via a single intramuscular injection in SD rats was 13.1&#xa0;μg/kg. In the sub-acute toxicity study, no adverse effects were observed up to the highest tested dose (6.0&#xa0;μg/kg/day), which was therefore considered the no observed adverse effect level (NOAEL) under the conditions.</p>