Medium-chain fatty acids (MCFAs) are an ideal feedstock for biodiesel and a range of oleochemical products. In this study, different combinations of CnFATB3, CnLPAAT-B and CnKASI from coconut (Cocos nucifera L.) were coexpressed in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system. Transgenic lines expressing different combinations of these genes were designated FL (FatB3 + LPAAT-B), FK (FatB3 + KASI) and FLK (FatB3 + LPAAT-B + KASI). The homozygous seeds of transgenic Arabidopsis thaliana expressing high levels of these genes were screened, and their fatty acid composition and lipid contents were determined. Compared with its content in wild-type A. thaliana, the lauric acid (C12:0) content was significantly increased by at least 395%, 134% and 124% in FLK, FL and FK seeds, respectively. Meanwhile, the myristic acid (C14:0) content was significantly increased by at least 383%, 106% and 102% in FL, FLK and FK seeds, respectively, compared to its level in wild-type seeds. Therefore, the FLK plants exhibited the best effects to increase the level of C12:0, and FL expressed the optimal combination of genes to increase the level of 14:0 MCFA.