Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. It was reported as the major mechanism of anticancer drug-induced cells death. Unfortunately, many of these drugs are non-specific and cause severe side effects. The effects of 5-fluorouracil (5-FU) on the apoptotic events in normal murine thymus were evaluated using an in vivo model. A single dose of 5-FU (150 mg/kg ip) was injected to CF-1 mice. A multiparametric analysis of thymic weight, cellularity, viability, architectural organization, apoptosis, DNA fragmentation, and the expression of several apoptotic proteins was evaluated in 10 days time-course study post-5-FU dosing. Total organ weights, thymocyte counts, and cell viabilities diminished drastically from the second day. The thymus architecture assessed through electron scanning microscopy revealed deep alterations and the lost of cell-cell contact between the first and the third days. DNA fragmentation and apoptotic indexes (May Grünwald Giemsa staining, double fluorescent dyes, and TdT-mediated dUTP nick-end labeling assay) revealed that cell death was maximal on the second day (three times over control). Furthermore, the pro-apoptotic proteins FAS and Bax were strongly up-regulated during the first 2 days. The aforementioned morphological and biochemical changes were also accompanied within the same period by caspase 3 activation. This study revealed that in vivo apoptosis in normal thymus after 5-FU administration is related to FAS, Bax, and Caspase 3 co-expressions under the current experimental conditions, these findings, therefore, contribute to a new insight into the molecular mechanisms involved during 5-FU administration upon the thymus and the possible events committed in the lymphophenia associated with chemotherapy.