Mammalian Pumilio proteins, PUM1 and PUM2, are members of the PUF family of sequence-specific RNA-binding proteins. In this review, we explore their mechanisms, regulatory networks, biological functions, and relevance to diseases. Pumilio proteins bind an extensive network of mRNAs and repress protein expression by inhibiting translation and promoting mRNA decay. Opposingly, in certain contexts, they can activate protein expression. Pumilio proteins also regulate noncoding (nc)RNAs. The ncRNA, ncRNA activated by DNA damage (NORAD), can in turn modulate Pumilio activity. Genetic analysis provides new insights into Pumilio protein function. They are essential for growth and development. They control diverse processes, including stem cell fate, and neurological functions, such as behavior and memory formation. Novel findings show that their dysfunction contributes to neurodegeneration, epilepsy, movement disorders, intellectual disability, infertility, and cancer. Mammalian Pumilio proteins recognize specific RNA sequences via a highly conserved Pum-homology domain (HD). Pumilio proteins bind and regulate a large number of RNAs. Pumilio proteins repress target mRNAs by antagonizing translation and promoting RNA degradation. In certain contexts, Pumilio proteins may activate gene expression. Pumilio proteins regulate stem cell fate, development, and neurological functions. Dysfunction of Pumilio proteins contributes to neurodegeneration, epilepsy, ataxia, infertility, and cancer.