We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura-) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura- strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker-gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.