Females of social Hymenoptera only mate at the beginning of their adult lives and produce offspring until their death. In most ant species, queens live for over a decade, indicating that ant queens can store large numbers of spermatozoa throughout their long lives. To reveal the prolonged sperm storage mechanisms, we identified enriched genes in the sperm-storage organ (spermatheca) relative to those in body samples in Crematogaster osakensis queens using the RNA-sequencing method. The genes encoding antioxidant enzymes, proteases, and extracellular matrix-related genes, and novel genes that have no similar sequences in the public databases were identified. We also performed differential expression analyses between the virgin and mated spermathecae or between the spermathecae at 1-week and 1-year after mating, to identify genes altered by the mating status or by the sperm storage period, respectively. Gene Ontology enrichment analyses suggested that antioxidant function is enhanced in the spermatheca at 1-week after mating compared with the virgin spermatheca and the spermatheca at 1-year after mating. In situ hybridization analyses of 128 selected contigs revealed that 12 contigs were particular to the spermatheca. These genes have never been reported in the reproductive organs of insect females, suggesting specialized roles in ant spermatheca.