DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin‐ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron‐containing and intron‐less amoebae are mated. The mobility process is fast, being completed in 5–10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron‐encoded protein was expressed in Escherichia coli and found to be an endonuclease, I‐Dir I, that cleaves an intron‐less ribosomal DNA allele at the intron‐insertion site, and is probably involved in intron homing. The endonuclease I‐Dir I seems to be a rare example of a protein that is expressed from a ribozyme‐processed RNA polymerase I transcript in vivo.