The functional maturation of human pancreatic β-cells remains poorly understood. EndoC-βH2 is a human β-cell line with a reversible immortalized phenotype. Removal of the two oncogenes, SV40LT and hTERT introduced for its propagation, stops proliferation, triggers cell size increase and senescence, promotes mitochondrial activity and amplifies several β-cell traits and functions. Overall, these events recapitulate several aspects of functional β-cell maturation. We report here that selective depletion of SV40LT, but not of hTERT, is sufficient to revert EndoC-βH2 immortalization. SV40LT inhibits the activity of the RB family members and of P53. In EndoC-βH2 cells, the knock-down of RB itself, and, to a lesser extent, of its relative P130, precludes most events triggered by SV40LT depletion. In contrast, the knock-down of P53 does not prevent reversion of immortalization. Thus, an increase in RB and P130 activity, but not in P53 activity, is required for functional maturation of EndoC-βH2 cells upon SV40LT-depletion. In addition, RB and/or P130 depletion in SV40LT-expressing EndoC-βH2 cells decreases cell size, stimulates proliferation, and decreases the expression of key β-cell genes. Thus, despite SV40LT expression, EndoC-βH2 cells have a residual RB activity, which when suppressed reverts them to a more immature phenotype. These results show that the expression and activity levels of RB family members, especially RB itself, regulate the maturation state of EndoC-βH2 cells.