Tristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3′ untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome. To assess the importance of the highly conserved C-terminal CNOT1 binding domain (CNBD) of TTP to the TTP deficiency phenotype in mice, we created a mouse model in which TTP lacked its CNBD. CNBD deletion mice exhibited a less severe phenotype than the complete TTP KO mice. In macrophages, the stabilization of target transcripts seen in KO mice was partially normalized in the CNBD deletion mice. In cell-free experiments, recombinant TTP lacking its CNBD could still activate target mRNA deadenylation by purified recombinant Schizosaccharomyces pombe CCR4/NOT complexes, although to a lesser extent than full-length TTP. Thus, TTP lacking its CNBD can still act to promote target mRNA instability in vitro and in vivo These data have implications for TTP family members throughout the eukarya, since species from all four kingdoms contain proteins with linked TZF and CNOT1 binding domains.