Phytosterols are a type of bioactive substances widely found in plants and have broad application prospects in food, medicine, cosmetics and other fields. Phytosterols, as cholesterol analogues, can inhibit the absorption of cholesterol in the intestine and decrease serum cholesterol level and thus reduce the risk of cardiovascular diseases. In addition, phytosterols have many other functions such as cancer suppression, anti-inflammation or anti-fever, anti-oxidation and hormone-like effects. In-depth exploration of the subcellular and molecular mechanism of phytosterols’ biological functions contributes to the full development of the application value of phytosterols. Mitochondria are the most important sites for cellular energy metabolism. Cholesterol metabolism, cancer cell proliferation and apoptosis, oxidative stress, and inflammatory response are all closely related to mitochondrial function. Recent studies have suggested that phytosterols can regulate mitochondrial function in various models, which potentially may be a pivotal mechanism underlying phytosterols’ various biological functions. This article will first summarize the biological functions of phytosterols and then discuss its mitochondria-related regulatory mechanisms in detail, hoping to provide frontier insights and progress report for researchers in the field as well as to provide reference for the application of phytosterols.