Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4′OMT transformants. HPLC/liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4′OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.