Simple sequence repeat-enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty-three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.