Now in its third decade of mechanistic investigation, testicular injury caused by 2,5-hexanedione (2,5-HD) exposure is a well-studied model with a rich database. The development of this model reflects the larger changes that have moved biology from a branch of chemistry into the molecular age. Critically examined in this review is the proposed mechanism for 2,5-HD-induced testicular injury in which germ cell maturation is disrupted owing to alterations in Sertoli cell microtubule-mediated functions. The goal is to evaluate the technical and conceptual approaches used to assess 2,5-HD-induced testicular injury, to highlight unanswered questions, and to identify fruitful avenues of future research.