In eukaryotic cells, deadenylation is achieved by deadenylases, which are 3"-5" exonuclease that specifically degrade poly(A) or oligo(A) at the 3"-end of RNAs. Most eukaryotic cells contain more than a dozen of deadenylase isoenzymes. Among them, the CCR4-NOT complex and the PAN2-PAN3 complex are the main contributors of non-specific deadenylation of mRNAs, while PARN and PNLDC1 are involved in highly regulated deadenylation of mRNAs and the biogenesis of non-coding RNAs. Besides their roles in RNA metabolism, deadenylases are also regulators of transcription, translation efficiency, stress response, immunological response, genome integrity, and self-renewal and differentiation of stem cells. In vitro and in vivo studies have discovered that deadenylase activity can be modulated by low-molecular-weight compounds, intramolecular interactions between catalytic and non-catalytic/structural domains, post-translational modifications, and binding partners. By regulating the 3"-tail length of poly(A) or oligo(A) of RNAs, deadenylases have been found to participate in diverse cellular, physiological and pathological processes by modulating RNA homeostasis. Particularly, deadenylases are key players of development by regulating the clearance of maternal mRNAs, the expression of tissue-specific genes and the cross-talk with developmental signaling pathways. Recently, inherited mutations or aberrant expression of deadenylases has been associated with many diseases including telomere diseases, cardiovascular diseases, neurodevelopmental diseases, cancers, and metabolic diseases. The precise regulation of deadenylases in their diverse intracellular functions may be achieved by a complicated network composing of various cis-acting elements in the targeted RNAs, thousands of trans-acting RNA-binding proteins, and numerous post-translational modifications. In this network, RNA-binding proteins may act as hubs to bind with targeted RNAs with specific cis-acting elements and to recruit a distinct deadenylase via protein-protein interactions, and thereby to modulate RNA fate by modifying the poly(A) length or trimming the oligo(A) at the 3"-end. The changes in the expression profile of RNA-binding proteins and in the post-translational modifications of deadenylase-binding partners provide a dynamic and responsive network to achieve the spatiotemporal regulation of gene expression. This complicated regulating network facilitates the cells to maintain RNA homeostasis or switch transcriptome to meet the demands of cell growth, proliferation, cell differentiation, stress response and cell death. The regulating network of deadenylases may also cross-talk with the other cellular pathways such as signaling transduction, autophagy, and anabolism of various biomacromolecules. In this review, we will discuss the regulators of deadenylases, the mechanisms of RNA homeostasis regulated by deadenylation, and the emerging roles of deadenylases in health and diseases.