We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable alpha-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alphaCP mRNA stability protein. Sequestration of alphaCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alphaCP to the alpha-globin 3'UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by alphaCP, implying that a stable higher-order structural network is involved in stabilization of the alpha-globin mRNA. Upon deadenylation, the interaction of PABP with alphaCP would be disrupted, rendering the alpha-globin 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.