Insulin-like growth factor II (IGF2) is a growth-promoting peptide that increases β-cell proliferation and survival. The aim of the study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. Islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3 [1.45% (IQR: 0.26) vs. 0.58% (IQR: 0.18), p = 0.006], 10 [1.58% (IQR: 1.40) vs. 0.90% (IQR: 0.61), p = 0.035], and 28 [1.35% (IQR: 0.35) vs. 0.64% (IQR: 0.28), p = 0.004] after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group [0.36 mg (IQR: 0.26) vs. 0.38 mg (IQR: 0.19)], it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group [0.63 mg (IQR: 0.38) vs. 0.42 mg (IQR: 0.31), p = 0.008]. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia.