The FOXO transcription factor DAF-16, a downstream effector of Insulin/IGF-1 signaling (IIS), is a critical longevity determinant in diverse organisms. However, the molecular basis of its regulation is still largely unknown. By mass spectrometry of affinity-purified DAF-16-associated factors, we have identified the deubiquitylating enzyme MATH-33 as a co-regulator for DAF-16 activity. We show that MATH-33 stabilizes DAF-16 protein levels in C. elegans under conditions when IIS is downregulated and DAF-16 is transcriptionally active. In addition, MATH-33 is required for maintaining DAF-16 function to regulate early developmental decisions and life span. Disruption of
math-33 expression in C. elegans significantly reduces DAF-16 protein levels under low IIS conditions without affecting
daf-16 mRNA levels. Genetic ablation of both
math-33 and
rle-1, a previously identified DAF-16 ubiquitin E3 ligase, is able to partially rescue DAF-16 activity, indicating an epistatic relationship between both factors for regulating DAF-16 levels. Finally, we demonstrate that expression of a catalytically inactive form of MATH-33 in cultured mammalian cells results in an increase of ubiquitylated DAF-16. Our data support a model in which MATH-33 acts as a deubiquitylating enzyme to stabilize DAF-16 protein levels when IIS is downregulated.