The use of magnetic nanoparticles (MNPs) magnetized on applying an alternating magnetic field (AMF) to stimulate the thermal characteristics and to induce tumor apoptosis is a currently active area of research in cancer treatment. In previous work, we developed biocompatible and superparamagnetic polystyrene-sulfonic-acid-coated magnetic nanoparticles (PSS-MNPs) as applications for magnetically labeled cell trapping, but without assessment of treatment effects on tumor diseases. In the present work, we examined PSS-MNP-induced magnetic fluid hyperthermia (MFH) on SK-Hep1 hepatocellular carcinoma (HCC) cells for lethal thermal effects with a self-made AMF system; an adjustable AMF frequency generated a variable intensity of magnetic field and induced MNP relaxation. The extracellular and intracellular MFH treatments on a SK-Hep1 cell line were implemented in vitro; the result indicates that the lethal effects were efficient and caused a significantly decreased cell viability of SK-Hep1 cells. As the PSS-MNP concentration decreased, especially in intracellular MFH treatments, the MFH effects on cells, however, largely decreased through heat spreading to the culture medium. On controlling and decreasing the volume of culture medium, the problem of heat spreading was solved. It can be consequently expected that PSS-MNPs would be a prospective agent for intracellular cancer magnetotherapy.