Methylobacterium extorquens AM1 is the model strain for methylotrophic bacteria that metabolize methanol as the sole carbon and energy source. Genetically modified M. extorquens AM1 is used as a methylotrophic cell factory (MeCF) for high value-added chemical production. We tested the Cre-loxP recombination system for its ability to mediate multicopy gene integration of the mvt3 operon (mvt3) in M. extorquens AM1. mvt3 controls the expression of the first three enzymes of the mevalonate synthesis pathway. We assayed for Cre-mediated multigene integration by screening for multicopy mutants via their survival in culture with a high kanamycin concentration (600 mu g/mL). We identified mutant strains in which the mevalonate titer was increased by up to 1.9-fold compared with M2 (M. extorquens AM1 Delta celABC Delta attTn7::mvt3::loxP) and confirmed mvt3 integration at 2-3 copies per genome. This result demonstrates the feasibility of multicopy integration in M. extorquens AM1 mediated by Cre-loxP recombination and its potential for improving the output of M. extorquens AM1 metabolic pathways, e.g., optimization of terpenoid synthesis.