Abstract Chronic lymphocytic leukemia (CLL) is characterized by chronic clonal expansion of mature CD19-expressing B lymphocytes and global dysfunction of immune effectors, including natural killer (NK) cells. CLL remains incurable, and novel approaches to refractory CLL are needed. Our group has previously described trispecific killer engager (TriKE) molecules that redirect NK cell function against tumor cells. TriKE reagents simultaneously bind an activating receptor on NK cells, CD16, and a tumor antigen while also providing an NK cell expansion signal via an interleukin-15 moiety. Here we developed the novel CD19-targeting 161519 TriKE. We demonstrate that 161519 TriKE induced killing of a CD19-expressing Burkitt’s lymphoma cell line and examined the impact on primary CLL targets using healthy donor and patient NK cells. 161519 TriKE induced potent healthy donor NK cell activation, proliferation, and directed killing. Furthermore, 161519 TriKE rescued the inflammatory function of NK cells obtained from CLL patient peripheral blood samples. Finally, we show that 161519 TriKE induced better directed killing of CLL in vitro when compared with rituximab. In conclusion, 161519 TriKE drives a potent activating and proliferative signal on NK cells, resulting in enhanced NK cell expansion and CLL target killing. Our findings indicate the potential immunotherapeutic value of 161519 TriKE in CLL.