Gastric cancer is one of the most common cancers in modern societies. Previous studies have shown that the use of nanoparticle complexes is effective in the treatment of cancer. The aim of this study was to investigate the cytotoxicity and anticancer properties of cobalt oxide (Co 3 O 4 ) nanoparticles (NPs) functionalized by glutamic acid (Glu) and conjugated with thiosemicarbazide (TSC) on gastric cancer (AGS) cell line. First, the Co 3 O 4 @Glu/TSC nanoparticles were synthesized via co-condensation reaction. Fourier-transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) tests were performed for identifying the morphology, structure, size and functional groups of produced nanoparticles. MTT assay was also performed to evaluate cytotoxicity effect. Moreover, Annexin V/PI staining with flow cytometry analysis, caspase-3 activation assay, and Hoechst 33258 staining was carried out for evaluating apoptosis. The FTIR results showed that the components of Co 3 O 4 @Glu/TSC NPs complex were successfully fabricated. Crystallographic structure of Co 3 O 4 @Glu/TSC NPs was confirmed by XRD patterns. SEM results indicated that the size of the nanoparticles was in the range of 16-40 nm. An EDX spectrum was determined and data explained the existence of cobalt as the prominent element. MTT test results showed that AGS cell life was significantly decreased compared to the control group with increasing concentration of nanoparticles (dose-dependent) (P < 0.05), IC 50 = 107.5 μg/mL. The results of flow cytometry assay and caspase-3 activity showed that fabricated Co 3 O 4 @Glu/TSC NPs induced apoptosis in the treated group. Moreover, Co 3 O 4 @Glu/TSC NPs treated AGS cells indicate an increase in the apoptotic characteristics including nuclear fragmentation. In the current work, the promising cytotoxicity and anti-cancer activities of Co 3 O 4 @Glu/TSC NPs complex toward gastric cancer (AGS) cell line were showed and it can be suggested for the drug delivery system.