Tetrahymena thermophila is a useful model for the study of eukaryotic biology. A neomycin resistance gene (neo) has been developed that was optimized for the codon usage of T. thermophila. Using this codon-optimized neo gene (neoTet), a new drug resistance marker cassette, neo4, has been constructed. The neo4 cassette resulted in about ten times more drug resistant transformants than a cassette containing the non-codon-optimized original neo gene. The new cassette enables transgenic Tetrahymena strains to be created with high efficiency. This study also emphasizes the importance of codon optimization in transgene expression in Tetrahymena.