Natural Killer (NK) cells represent a cytotoxic group of cells that play important roles in immune surveillance and antitumor activities. They mediate the death of target cells through activating receptors in a Human Leukocyte Antigen (HLA)-unrestricted manner. NK cells can be obtained from a wide variety of allogeneic sources, opening roads for the development of a well-characterized and off-the-shelf product. As a result, these cells have become an attractive source for cancer immunotherapy. The most recent application of NK cells in immunotherapy is through the generation of CAR-NK cells, which are redirected to specific cancer epitopes. The co-stimulatory region of CARs used to generate CAR-NK cells are primarily derived from those developed to activate T-cells, therefore, it may not be well suitable to properly activate NK cells to make them exert their full cytotoxic potential. In this context, we developed new CAR constructs combining in their stimulatory regions molecules that naturally are involved in NK activation and compared with the commonly used 4-1BB-CD3ζ based CAR, aiming to improve effector functions of CAR-NK cells. It is described the capacity of Tyrosine Kinase Inhibitors (TKI) to improve NK cells cytotoxic function, therefore, in combination with the new CARs, we assessed the treatment of CAR-NK cells with the TKI Dasatinib to enhance CAR-NK effector function in the context of antitumor activity. NK-92 cells were lentivirally transduced with viral particles containing the coding sequences of 4th generation anti-CD19 CARs, named: A (4-1BB-CD3ζ), B, C and D. Then, the CAR+ cells were FACS-sorted based on the same fluorescence intensity in order to generate populations with equal CAR presence on the cell membrane. Next, cytotoxicity, degranulation and cytokine production were assessed in co-culture assays against CD19-expressing Nalm-6 or Raji (leukemia and lymphoma, respectively) cell lines. For Dasatinib treatment, the CAR-NK cells were incubated for 24 hours with 10nM of the inhibitor or DMSO as control and then the cells were washed and co-culture assays were performed against Nalm-6 and Raji cells. Using flow cytometry based cytotoxicity assay, CAR-NK cells demonstrated higher killing capacity against either Nalm-6 and Raji cells in all different effector:target ratios, compared with non-transduced NK-92 cells. This higher cytotoxicity was corroborated by the increase in CD107a detection on the cell surface of CAR-NK cells (degranulation). This robust cell activation resulted in higher production of the pro-inflammatory cytokines IFN-γ and TNF-α compared with non-transduced cells. The effector functions against Nalm-6 performed by the cells carrying the CARs C and D demonstrated to be superior compared with CARs A and B. However, when the target was Raji cells, there were no differences between the constructions. Strikingly, the treatment of the CAR-NK cells with Dasatinib enhanced almost 40% in cell cytotoxicity against Raji cells. Interestingly, considering assays against Nalm-6 cells, only the cells expressing CARs C and D demonstrated enhancement in effector functions, probably due the differences in the activation domains, with an augment of about 50% in cell cytotoxicity, and higher degranulation and cytokine production compared to cells expressing CARs A and B. Taken together, these findings indicate that the use of NK-specific molecules in the CAR stimulatory domain augments the effector function potency of NK-92 cells. Furthermore, the utilization of the Src kinase inhibitor Dasatinib appears to further amplify the antitumor activity of CAR-NK cells in a CAR-specific manner.