Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, has emerged as a significant biomarker in the study of various pathological conditions. Dysregulation of homocysteine metabolism has been linked to cardiovascular diseases, neurological disorders, and developmental abnormalities. This review aims to provide an overview of the current understanding of the role of cofactors in homocysteine metabolism, highlighting the mechanisms by which they modulate enzyme activity, and their implications in health and disease. A comprehensive search of scientific literature was performed to identify relevant studies investigating the role of cofactors in homocysteine metabolism. Studies encompassing both in vitro experiments and clinical observations were included. The selected studies were critically evaluated to elucidate the molecular mechanisms by which cofactors influence key enzymes involved in homocysteine metabolism. In vitro studies have consistently demonstrated the essentiality of cofactors, such as folate, vitamin B12, and vitamin B6, in the proper functioning of enzymes that regulate homocysteine metabolism. Folate, in the form of 5-methyltetrahydrofolate, acts as a methyl donor for the remethylation of homocysteine to methionine via methionine synthase. Vitamin B12 serves as a cofactor for methionine synthase and methionine synthase reductase, facilitating the conversion of homocysteine to methionine. Vitamin B6, as pyridoxal phosphate, is involved in the transsulfuration pathway, converting homocysteine to cysteine through the action of cystathionine beta-synthase. Dysregulation or deficiencies in these cofactors disrupt the enzymatic activity of these key enzymes, leading to impaired homocysteine metabolism and elevated homocysteine levels. The reviewed literature underscores the critical role of cofactors in homocysteine metabolism. Adequate levels of folate, vitamin B12, and vitamin B6 are essential for the proper functioning of enzymes involved in homocysteine clearance pathways. Deficiencies in these cofactors have been consistently associated with elevated homocysteine levels, which have been implicated in the pathogenesis of numerous diseases. Consequently, maintaining optimal levels of these cofactors through dietary intake or supplementation holds promise as a potential therapeutic strategy for preventing or managing homocysteine-related disorders. However, further research is needed to elucidate the optimal dosage, duration, and potential interactions with other factors to maximize the benefits of cofactor supplementation.