Lamina-associated polypeptide (LAP) 2α is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2α in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2α by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2α-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2α COOH terminus directly bound Rb, and overexpressed LAP2α inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2α associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2α in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2α on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.