Thiosemicarbazones (TSCs), and their copper derivatives, have been extensively studied mainly due to the potential applications as antitumor compounds. A part of the biological activity of the TSC-Cu complexes rests on their reactivity against cell reductants, as glutathione (GSH). The present paper describes the structure of the [Cu(PTSC)(ONO)] compound (1) (HPTSC=pyridine-2-carbaldehyde thiosemicarbazone) and its spectroscopic and magnetic properties. ESI studies performed on the reaction of GSH with 1 and the analogous [{Cu(PTSC*)(ONO)}] derivative (2, HPTSC*=pyridine-2-carbaldehyde 4N-methylthiosemicarbazone) show the absence of peaks related with TSC-Cu-GSH species. However GSH-Cu ones are detected, in good agreement with the release of Cu ions after reduction in the experimental conditions. The reactivity of 1 and 2 with cytochrome c and myoglobin and their activities against HT-29 and SW-480 colon carcinoma cell lines are compared with those shown by the free HPTSC and HPTSC* ligands.