Cypermethrin (CP) is a neurotoxic insecticide found accumulated in oysters, one of the most commonly consumed seafoods, posing potential health risks to the human body. We designed a gastrointestinal tracing method allowing for accurate quantification of the propulsion of chyme and further established the mouse in vivo digestion model to explore the behavior of CP in the digestion of raw, steamed, and roasted oysters. The results showed that bioaccumulation of CP in oysters may be accompanied by the biotransformation of CP. Thermal processing decreased both the CP content in oysters and its bioaccessibility. The small intestine is the main site for CP digestion and absorption. The cis-isomers of CP might finally accumulate in the body at a higher ratio and further become the predominant configuration for toxic effects. Taken together, the study contributes to the risk assessment of the dietary exposure of CP from aquatic products.