The 24-h molecular clock is based on the stability of rhythmically expressed transcripts. The shortening of the poly(A) tail of mRNAs is often the first and rate-limiting step that determines the lifespan of a mRNA and is catalyzed by deadenylases. Herein, we determine the catalytic site of Hesperin, a recently described circadian deadenylase in plants, using a modified site-directed mutagenesis protocol and a custom vector, pATHRA. To explore the catalytic efficiency of AtHESPERIN, we investigated the effect of AMP and neomycin, and used molecular modeling simulations to propose a catalytic mechanism. Collectively, the biochemical and in silico results classify AtHESPERIN in the exonuclease-endonuclease-phosphatase deadenylase superfamily and contribute to the understanding of the intricate mechanisms of circadian mRNA turnover.