Piwi-interacting RNAs (piRNAs) are small non-coding RNAs that play a crucial role in gene regulation and immune defense. This study investigates their function in Penaeus vannamei shrimp during White Spot Syndrome Virus (WSSV) infection. Analysis of small RNA libraries from WSSV-infected shrimp hemocytes identified 82,788 piRNA homologs, with 138 showing altered expression during infection. Putative piRNAs were mapped to both the P. vannamei nuclear and mitochondrial genomes, highlighting their diverse origins. Interestingly, some piRNA sequences from uninfected shrimp mapped to both the shrimp and WSSV genomes, suggesting potential subversion or integration of viral fragments into the host genome. We focused on piR-pva-926938, a downregulated piRNA targeting the WSSV186 gene. Introducing piR-pva-926938 into WSSV-infected shrimp suppressed WSSV186 expression, but paradoxically increased viral load by downregulating host immune genes like calcineurin B and dynamin-binding protein. This study is the first to report WSSV-responsive piRNAs in shrimp and reveals the complex interplay between piRNAs, viral genes, and host immunity during WSSV infection.