This study reports the synthesis of a number of 1- and 2-phenyl derivatives of the 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole nucleus, which were obtained by the reaction of the versatile 7-substituted 2,3-dihydro-3-hydroxymethylene-4H-1-benzothiopyran-4-ones with hydrazine and substituted phenylhydrazines. The antiproliferative activity of the synthesized compounds was evaluated by an in vitro assay on human tumor cell lines (HL-60 and HeLa) and showed a significant capacity of the 7-methoxy-substituted benzothiopyrano[4,3-c]pyrazoles 3b-d, carrying the pendant phenyl group in the 1-position, to inhibit cell growth. Investigation of the mechanism of action indicated the induction of the mitochondrial permeability transition (MPT) as the molecular event responsible for the inhibition of cell growth. This phenomenon is related to the ability of the test compounds to cause a rapid Ca2+-dependent and cyclosporin A-sensitive collapse of the transmembrane potential (DeltaPsi) and matrix swelling. All this leads to the release of caspase activators, such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF), which trigger the pro-apoptotic pathway leading to DNA fragmentation.