4,4-Dimethyl phytosterols possess two methyl groups at the carbon-4 atom of the aliphatic A-ring. The methyl groups are crucial for the molecular recognition of endogenous and exogenous bioactive compounds. Phytosterols have received worldwide attention owing to their recognized health benefits. However, 4,4-dimethyl phytosterols are less appreciated. Recent research studies revealed that 4,4-dimethyl phytosterols exert numerous beneficial effects on disease prevention, and are particularly involved in the endogenous cannabinoid system (ECS). The purpose of this review is to summarize and highlight the currently available information regarding the structures and sources of 4,4-dimethyl phytosterols, and to provide detailed preclinical studies performed to evaluate their potential for treating various diseases. Future research on 4,4-dimethyl phytosterols is warranted to confirm their relationship with the ECS, and to elucidate the mechanism directly toward clinical trials.