Background <p>NK cells engineered to express interleukin-15 (IL-15) and a CD19-targeted chimeric antigen receptor (CAR) have been used to treat patients with relapsed and/or refractory B cell malignances, demonstrating encouraging outcomes and favorable safety profile. However, the effect of IL-21 in CAR-NK cell therapy remains unknown.</p> Methods <p>CD19-specific CAR with 4-1BB costimulatory domain and cytokine IL-21 or IL-15 was constructed and transduced into peripheral blood (PB)-derived NK cells to produce CD19-CAR-IL21 NK cells (CAR-21) or CD19-CAR-IL15 NK cells (CAR-15), respectively. The phenotypic profile, transcriptomic characteristics, functionality and anti-tumor activity of CAR-21 NK cells and CAR-15 NK cells were compared.</p> Results <p>Compared with CAR-NK cells co-expressing IL-15, CAR-NK cells co-expressing IL-21 exhibited significantly increased IFN-γ, TNF-α and Granzyme B production, as well as degranulation, in response to CD19⁺ Raji lymphoma cells, resulting in enhanced cytotoxic activity upon repetitive tumor stimulation. Furthermore, IL-21 co-expression improved the in vivo persistence of CAR-NK cells and significantly suppressed tumor growth in a xenograft Raji lymphoma murine model, leading to prolonged survival of CD19⁺ tumor-bearing mice. RNA sequencing revealed that CAR-21 NK cells have a distinct transcriptomic signature characterized by enriched in cytokine, cytotoxicity, and metabolic related signaling, when compared with CAR-15 NK or CAR NK cells.</p> Conclusions <p>This study demonstrated that CD19-specific CAR-NK cells engineered to express IL-21 exhibit superior persistence and anti-tumor activity against CD19⁺ tumor compared to CAR-NK cells co-expressing IL-15, which might be a promising therapeutic strategy for treating patients with relapse or refractory B cell malignances.</p>