The Streptomyces coelicolor wblA gene is known to play a negative role in both antibiotic biosynthesis and the expression of genes responding to oxidative stress. Recently, WhcA, a WblA ortholog protein, was confirmed to interact with dioxygenase-encoding SpiA (stress protein interacting with WhcA) in Corynebacterium glutamicum. We describe here the identification of a SpiA ortholog SCO2553 protein (SpiAsc) that interacts with WblA in S. coelicolor. Using heterologous expression in E. coli and in vitro pull-down assays, we show that WblA specifically binds SpiAsc, and is influenced by oxidants such as diamide. These data indicate that the interaction between WblA and SpiAsc is not only specific but also modulated by the redox status of the cell. Moreover, a spiAsc-disruption mutant exhibited a less sensitive response to the oxidative stress induced by diamide present in solid plate culture. Real-time RT-PCR analysis also showed that transcription levels of oxidative stress response genes (sodF, sodF2, and trxB) were higher in the spiAsc-deletion mutant than in wild-type S. coelicolor. These results show that SpiAsc negatively regulates WblA during oxidative stress responses in S. coelicolor.