Summary: The sea cucumber Apostichopus japonicus, a key species in Chinese aquaculture, plays a significant evolutionary role within the Echinodermata phylum. However, the sex determination mechanism in this species remains poorly understood. Here, we conducted extensive sex surveys and sampling of eight wild populations, investigating the sex-related SNPs and insertion or deletions (indels) through bulk segregation analysis (BSA) and genome-wide association study (GWAS) analysis. Our findings suggest that A. japonicus employs a polygenic sex determination (PSD) system, with solute carrier family 8 (SLC8A) being the candidate gene for sex determination, encoding sodium-calcium exchanger (NCX1). The analysis of normalized sequencing depth reveals variations across chromosomes 6, 13, 14, 16, and 18, supporting the PSD system. We also identified 541.656 kb of male-specific sequences and screened five markers (C77185, C98086, C64977, C125, and C876) for molecular sex identification. Overall, this study provides new insights into A. japonicus sex determination, highlighting a complex multi-gene mechanism rather than a simple XX/XY system.