Four novel gold(III) complexes of general formulae AuCl2{(S,S)-R2eddl}PF6 (R2eddl=O,O′-dialkyl-(S,S)-ethylenediamine-N,N′-di-2-(4-methyl)pentanoate, R=n-Pr, n-Bu, n-Pe, i-Bu; 1–4, respectively), were synthesized and characterized by elemental analysis, UV/Vis, IR, and NMR spectroscopy, as well as high resolution mass spectrometry. Density functional theory calculations pointed out that (R,R)-N,N′-configuration diastereoisomers were energetically the most favorable. Duo to high cytotoxic activity complex 3 was chosen for stability study in DMSO, no decomposition occurs within 24h, and for the reaction with ascorbic acid in which was reduced immediately. Additionally, 3 interacts with bovine serum albumin (BSA) as proven by UV/Vis spectroscopy. In vitro antitumor activity was determined against human cervix adenocarcinoma (HeLa), human myelogenous leukemia (K562), and human melanoma (Fem-x) cancer cell lines, as well as against non-cancerous human embryonic lung fibroblast cells MRC-5. The highest activity was observed against K562 cells (IC50: 5.04–6.51μM). Selectivity indices showed that these complexes are less toxic than cisplatin. 3 had a similar viability kinetics on HeLa cells as cisplatin. Drug accumulation studies in HeLa cells showed that the total gold uptake increased much faster than that of cisplatin pointing out that 3 more efficiently enters the cells than cisplatin. Furthermore, morphological and cell cycle analysis reveal that gold(III) complexes induced apoptosis in time- and dose-dependent manner. Synthesis, characterization, stability, interactions with bovine serum albumin and ascorbic acid, in vitro anticancer activity, cell cycle and mode of cell death, as well as kinetics study and drug uptake of novel gold(III) complexes of general formula AuCl2{(S,S)-R2eddl}PF6, (R2eddl=O,O′-dialkyl-(S,S)-ethylenediamine-N,N′-di-2-(4-methyl)pentanoate; R=n-Pr, n-Bu, n-Pe, i-Bu) are presented. Display omitted •Four new gold(III) complexes with N,N′-ethylenediamine bidentate esters are prepared.•Highest activity is against K562 cells, indicating novel antileukemic treatments.•The most active Au(III) complex accumulated in cells more efficiently than cisplatin.•The mode of cell death induced by these complexes is apoptosis.•The most active Au(III) complex interacts with bovine serum albumin (BSA).