MicroRNAs (miRNAs) regulate gene expression post-transcriptionally through binding specific sites within the 3' untranslated regions (UTRs) of their target mRNAs. Numerous investigations have documented repressive effects of miRNAs and identified factors required for their activity. However, the precise mechanisms by which miRNAs modulate gene expression are still obscure. Here, we have examined the effects of multiple miRNAs on diverse target transcripts containing artificial or naturally occurring 3' UTRs in human cell culture. In agreement with previous studies, we report that both the 5' m(7)G cap and 3' poly(A) tail are essential for maximum miRNA repression. These cis-acting elements also conferred miRNA susceptibility to target mRNAs translating under the control of viral- and eukaryotic mRNA-derived 5' UTR structures that enable cap-independent translation. Additionally, we evaluated a role for the poly(A)-binding protein (PABP) in miRNA function utilizing multiple approaches to modulate levels of active PABP in cells. PABP expression and activity inversely correlated with the strength of miRNA silencing, in part due to antagonism of target mRNA deadenylation. Together, these findings further define the cis- and trans-acting factors that modulate miRNA efficacy.