The thymus is the primary site of T cell development, central to the establishment of self-tolerance and adaptive immune function. In mammals, the thymus undergoes age-related involution, resulting in a global decline in immune function. The thymus has some regenerative ability that relies on pre-existing thymic remnants but is insufficient to prevent involution. Here, we show that the juvenile axolotl (Ambystoma mexicanum) is able to regenerate its thymus de novo after complete removal, constituting an exception among vertebrates. Using single-cell transcriptomics and genetic and transplantation approaches, we demonstrate that de novo thymus regeneration results in the restoration of morphology, cell-type diversity, and function. FOXN1, although it has a conserved role in thymus organogenesis, is dispensable for the initiation of thymic regeneration. In contrast, we identify midkine signaling as a possible early driver of de novo thymus regeneration. This study demonstrates an instance of organ-level regeneration of the lymphoid system, which could guide future clinical strategies seeking to promote thymus regrowth.