Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2β) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2β has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2β transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.