The objective of this study was to investigate the role of the replacing animal fat in the meat gel system with lard-based diacylglycerol (DG), which has health benefits and similar characteristics (such as taste, smell, and usability) to lard triacylglycerol (TG, the major component of pork fat). The impact of lard-based DG on the thermal gelation and rheological properties of myofibrillar protein (MP) as influenced by the incorporation of soy protein isolate (SPI, 0–0.75% protein) was investigated, and the interaction mechanism was also explored. The gel structure became more continuous and compact with the incorporation of DG or purified DG (PDG) and the decrease of SPI content, as reflected by the change of secondary structures, which was beneficial to restrict the mobility of immobilized water. With the incorporation of DG or PDG and the decrease in the amount of SPI, the gel strength, G′, and G″ were increased. Molecular docking results further revealed that the DG interacted with SPI mainly through van der Waals forces and hydrogen bonds, and interacted with MP mainly through van der Waals forces, hydrogen bonds, and hydrophobic interactions. More importantly, the negative binding energies of the DG-protein complexes were lower than those of the TG-protein complexes, indicating that the interaction was spontaneous and that DG is more likely to interact with proteins than TG. In conclusion, this study provides a vital theoretical basis at the molecular level for the development of desired quality meat products containing DG with health benefits.