Integration of viral DNA into the host genome is an essential step in retroviral replication. The viral DNA made by reverse transcription is a component of the preintegration complex (PIC) that also contains the viral integrase protein, the enzyme that integrates the viral DNA. Several other viral and cellular proteins are present in the PIC, but their functional roles are less well established. Barrier-to-autointegration factor (BAF) is a cellular protein component of the PIC that blocks autointegration of the viral DNA and stimulates intermolecular integration. In uninfected cells, BAF interacts with members of the LEM family of inner nuclear membrane and nucleoplasmic proteins. Here, we demonstrate that one of the LEM proteins, lamina-associated polypeptide 2alpha (LAP2alpha), is a component of the PIC. LAP2alpha stabilizes the association of BAF with the PIC to stimulate intermolecular integration and suppress autointegration. To further understand the role of LAP2alpha, we established LAP2alpha-knockdown cell lines. Depletion of LAP2alpha significantly inhibited viral replication. Our results demonstrate a critical contribution of LAP2alpha to the nucleoprotein organization of the PIC and to viral replication.