Toxicological information on moniliformin (MON), an emerging mycotoxin, is limited. This study examined the acute and 28-day toxicity of orally administered MON in male ICR mice. Regarding the acute toxicity, among single oral doses of 0, 20, 40, and 80 mg/kg body weight (BW), MON caused proximal tubular necrosis in the kidneys at ≥ 40 mg/kg BW, and the lethal dose 50 value was estimated as 68.1 mg/kg BW. Regarding the 28-day toxicity, among oral doses of 0, 10, 20, and 40 mg/kg BW/day, MON increased absolute heart weight at 40 mg/kg BW, but histopathological changes were not evident in the heart. In contrast, 40 mg/kg BW MON induced centrilobular liver cell hypertrophy accompanied by increased absolute liver weight. Moreover, MON dose-dependently increased the absolute kidney weight at ≥ 20 mg/kg BW and increased the incidence of renal tubular regeneration at 40 mg/kg BW. RNA sequencing analysis in the renal cortex after a single dose of 40 mg/kg BW MON revealed upregulation of metabolic response-related genes, such as Cyp3a13, Cyp26b1, and Cyp4f15, and oxidative stress-related Gpx7. These results suggest that MON targets the kidneys in mice. Orally ingested MON may be metabolized in the kidneys as well as in the liver, and active intermediates or reactive oxygen species may induce renal tubular toxicity, causing proximal tubular necrosis. Based on kidney changes, the no-observed-adverse-effect-level of MON in the 28-day oral toxicity study of male mice was determined to be 10 mg/kg BW/day.