This study aims to evaluate and compare the cytotoxicity and biocompatibility of a modified heat-cured acrylic denture base material containing 15% phosphoric acid 2-hydroxyethyl methacrylate ester (PA2HEME) with those of nonmodified PMMA. Discs with a diameter of 12 mm and a thickness of 2 mm were prepared using a heat-cured PMMA denture base material and divided into control and experimental groups. The experimental group was modified with 15% phosphoric acid 2-hydroxyethyl methacrylate ester (PA2HEME). The modified and nonmodified materials were tested via FTIR, and the effect of modification on surface roughness was evaluated with AFM. An in vitro test was conducted to examine the cytotoxicity and biocompatibility of heat-cured acrylic denture base materials (control and experimental groups) by using a rat embryonic fibroblast cell line (REF). Cell culture viability was estimated with a methyltetrazolium solution cytotoxicity assay. The surface roughness of PA2HEME-modified acrylic resin (mPMMA) was not significantly different from that of nonmodified PMMA acrylic resin. The cytotoxicity test on mPMMA demonstrated no significant change in the cell viability compared with that of the negative control. No deteriorating or inhibitory effect on cell growth was observed after 24 and 72 h. The modification of denture base materials with 15% PA2HEME has no cytotoxic or inhibitory effects on the growth of the studied fibroblast cell line.