Stem-cell antigen 1–positive (Sca-1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′-azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β-arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β-arrestin2 in Sca-1+ CSC differentiation, we used β-arrestin2–knockout mice and overexpression strategies. Real-time PCR revealed that β-arrestin2 promoted 5′-azacytizine-induced Sca-1+ CSC differentiation in vitro. Because the microRNA 155 (miR-155) may regulate β-arrestin2 expression, we detected its role and relationship with β-arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR-155. Real-time PCR revealed that miR-155, inhibited by β-arrestin2, impaired 5′-azacytizine-induced Sca-1+ CSC differentiation. On luciferase report assay, miR-155 could inhibit the activity of β-arrestin2 and GSK3β, which suggests a loop pathway between miR-155 and β-arrestin2. Furthermore, β-arrestin2-knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β-arrestin2-Knockout mice, so the activity of GSK3β was regulated by β-arrestin2 not Akt. We transplanted Sca-1+ CSCs from β-arrestin2-knockout mice to mice with myocardial infarction and found similar protective functions as in wild-type mice but impaired arterial elastance. Furthermore, low level of β-arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β-arrestin2/miR-155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.