(1) Background: Acetamiprid, a widely used neonicotinoid insecticide, has increasingly drawn people’s attention to its potential reproductive toxicity risks. Although existing studies have shown that exposure to acetamiprid can cause testicular damage, its underlying molecular mechanism has not been fully elucidated. (2) Methods: This study established a mouse model of acetamiprid exposure via free drinking water and combined it with in vitro culture of TM3 cells, aiming to explore the toxic effects of acetamiprid on testicular tissue and its potential mechanism. (3) Results: Acetamiprid exposure induced significant pathological damage to the testicular tissue of mice and significantly reduced sperm quality. Further mechanism studies have shown that acetamiprid down-regulates the expression of RNA demethylase ALKBH5, leading to an overall increase in m⁶A modification levels, thereby inhibiting the activity of the PI3K/AKT signaling pathway and ultimately inducing apoptosis of TM3 cells. (4) Conclusions: In conclusion, this study, by integrating both internal and external models, has revealed a molecular mechanism by which acetamiprid is involved in testicular injury through epigenetic pathways, providing scientific basis and mechanism clues for assessing reproductive hazards caused by exposure to environmental toxins.