<p>The cytochrome P4503A (<i>CYP3A</i>) subfamily has been well characterized in vertebrates, where it plays a pivotal role in xenobiotic detoxification and the regulation of steroid hormone levels. However, their molecular features and functions in invertebrates remain poorly understood. The sea cucumber (<i>Apostichopus japonicus</i>) is not only a commercially significant mariculture species but also an excellent model for studying organ development and evolutionary biology. In this study, we identified seven <i>CYP3A</i> genes in <i>A. japonicus</i> using gonadal transcriptomic data. These genes exhibited ubiquitous expression across adult tissues, with significantly higher levels in ovarian tissues compared to testes tissues. Protein sequence analysis revealed that the catalytic residues Ala ₃₀₆-Thr ₃₁₀-Cys ₄₃₇, which are critical for aromatase activity, are highly conserved. This suggests that CYP3A proteins may mediate aromatization-like reactions similar to CYP19A in vertebrates. Additionally, dietary administration of the aromatase inhibitor letrozole led to histological changes in the testes and a reduction in the diameter of large oocytes in the ovaries. These findings provide valuable insights into the molecular mechanisms underlying gonadal development in sea cucumbers and lay the groundwork for future research on the artificial induction of sex reversal in echinoderms.</p>