Thyroid hormones (THs), and more precisely the 3,3′,5-triiodo-l-thyronine (T3) acetic derivative 3,3′,5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T3 and l-thyroxine (T4), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T3, suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage.