Here, we investigated antioxidant defense mechanism in the spermatheca of A. mellifera queens via RNA-seq analysis of spermathecae in both mated and virgin queens. We identified the genes encoding antioxidant proteins, which were differentially expressed in the spermatheca of mated queens. The concentrations of antioxidant proteins, such as superoxide dismutase 1 (SOD1), catalase, glutathione peroxidase (GTPX), and transferrin (Tf) together with the levels of ROS, H2O2, and iron were higher in the spermathecal fluid of mated queens as opposed to those in the spermathecal fluid of virgin queens; this indicated that increase in antioxidant protein concentration is an antioxidant defense mechanism occurring in the spermathecal fluid of mated queens against ROS; this mechanism involves conversion of ROS using antioxidant enzymes and Tf-mediated inhibition of the Fenton reaction occurring between Fe2+ and H2O2. Our data indicate that an increased expression of antioxidant proteins could facilitate prolonged storage and survival of sperms in the spermatheca of mated queens, suggesting the role of antioxidant proteins in antioxidative defense against ROS.