Aim. Development of effective transfection agents remains an actual topic in biotechnology. The human embryonic kidney (HEK-293) cell line is commonly used for transfections, including the large-scale format. Previously [1, 2], we have found that cationic calixarenes with choline or N-methylimidazolium groups condense DNA in nano-sized particles and promote transfection of HeLa and Cos-7 cells. This study includes the toxicity and transfection experiments on HEK 293 cells with calixarenes modified with choline, N-methylimidazolium, 3-pyridinium, aminoammonium (CXN), and amidoammonium (CXN3FAc) groups at the upper macrocycle rim and long alkyl chains (C8, C12 and C16) at the lower rim. Conclusions. The transfection efficiencies of calix[4]arenes CX8N and CX16N3FAc for the commonly used HEK293 cells were comparable to the commercial transfection reagent control. This contrasts with the low results for CX8N obtained previously on HeLa and Cos-7 cells. At the used N/P=5 ratio of calixarenes to DNA, their working concentrations were 3.7 µM, which is well below their cytotoxicity IC₅₀s. Therefore, these compounds are promising agents for the transfection formulations.