β-thymosins, including thymosin β4 (Tβ4), Tβ10, and Tβ15, are a family of highly conserved 5 kDa peptides. They are involved not only in normal cell migration, but also in tumor metastasis. However, the molecular mechanisms of β-thymosins to regulate cell migration and other functions are not fully understood. Recently, this important area is under active investigation worldwide. Many new discoveries have been made from molecular biology and cell culture models as well as animal models and human diseases. This timely review provides the most updated information about functional roles and molecular mechanisms of β-thymosins in normal tissues and disease conditions.