Methylmercury (MeHg) is one of the most toxic environmental pollutants and causes serious health hazards worldwide. Recently, we demonstrated that oleanolic acid (OA) 3-glucoside (OA3Glu), a saponin derivative in which glucose is bound to the C3 position of OA, has anti-MeHg activity by suppressing Hg accumulation in organs of mice. In this study, we examined the anti-MeHg activity of OA-3-(1′2′orthoacetate-Glu)-28-Glu in which glucose is bound to the C3 position of OA in a different binding form from that in OA3Glu. We found that OA-3-(1′2′orthoacetate-Glu)-28-Glu suppressed cellular MeHg uptake and improved cell viability upon exposure to MeHg in Caco-2 cells. To verify the in vivo anti-MeHg activities of OA-3-(1′2′orthoacetate-Glu)-28-Glu, mice were orally administered MeHg (0.02, 1.0, or 5.0 mg·kg⁻¹·d⁻¹), with or without OA-3-(1′2′orthoacetate-Glu)-28-Glu. The mice cotreated with 0.02 mg·kg⁻¹·d⁻¹ MeHg and OA-3-(1′2′orthoacetate-Glu)-28-Glu showed significantly lower Hg content in the liver and kidney than those treated with MeHg alone. In addition, interleukin (IL)-1β and IL-6 levels in the brain of mice cotreated with 5.0 mg·kg⁻¹·d⁻¹ MeHg and OA-3-(1′2′orthoacetate-Glu)-28-Glu were significantly lower than those of mice treated with MeHg alone. These results suggested that OA-3-(1′2′orthoacetate-Glu)-28-Glu had potential as an anti-MeHg accumulation compound, owing to its ability to suppress MeHg distribution into organs especially under low-level MeHg exposure condition. Taken together, it was suggested that glucose binding to the C3 position of OA is important for anti-MeHg activity of OA saponin derivatives.