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ABSTRACT Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.