The glycerol 3-phosphate shuttle (GPS) consists of two enzymes: cytosolic NAD⁺-linked glycerol 3-phosphate dehydrogenase 1 (GPD1) and mitochondrial FAD-linked glycerol 3-phosphate dehydrogenase 2 (GPD2). These enzymes play crucial roles in mitochondrial bioenergetics and the interplay between glucose and lipid metabolism. Abnormal expression of these genes has been linked to various metabolic diseases and cancers. This review summarizes the current understanding of the functions and effects of GPS, focusing on their involvement in diseases such as cancer. It also discusses the potential and challenges of developing therapeutic strategies targeting these enzymes. GPD1 is involved in the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) using NADH, and its activity is regulated at both transcriptional and post-translational levels. GPD1 has been associated with obesity, hyperlipidemia, and liver steatosis, but its exact role in human obesity remains unclear. GPD1L, a related protein, is associated with Brugada syndrome and sudden cardiac death. GPD2 is highly expressed in brown adipose tissue and plays a significant role in energy production and thermogenesis. It is regulated by multiple factors, including transcriptional and post-translational mechanisms. GPD2 has been linked to diabetes, muscle regeneration, and ischemic diseases. Overexpression of GPD2 can enhance glucose oxidation and support acetyl-CoA production, which may contribute to cancer growth. In cancer, GPD1 generally acts as a tumor suppressor, while GPD2 acts as a tumor promoter. GPD1 is often downregulated in cancer tissues, and its overexpression can inhibit cancer cell growth. GPD2 is upregulated in many cancer types and is correlated with poorer survival in some cases. GPD2 regulates cancer progression through various mechanisms, including ROS generation, bioenergetics, and ether lipid biosynthesis. Overall, the review highlights the complex roles of GPD1 and GPD2 in metabolic diseases and cancer, emphasizing the need for further research to fully understand their functions and develop effective therapeutic strategies.The glycerol 3-phosphate shuttle (GPS) consists of two enzymes: cytosolic NAD⁺-linked glycerol 3-phosphate dehydrogenase 1 (GPD1) and mitochondrial FAD-linked glycerol 3-phosphate dehydrogenase 2 (GPD2). These enzymes play crucial roles in mitochondrial bioenergetics and the interplay between glucose and lipid metabolism. Abnormal expression of these genes has been linked to various metabolic diseases and cancers. This review summarizes the current understanding of the functions and effects of GPS, focusing on their involvement in diseases such as cancer. It also discusses the potential and challenges of developing therapeutic strategies targeting these enzymes. GPD1 is involved in the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) using NADH, and its activity is regulated at both transcriptional and post-translational levels. GPD1 has been associated with obesity, hyperlipidemia, and liver steatosis, but its exact role in human obesity remains unclear. GPD1L, a related protein, is associated with Brugada syndrome and sudden cardiac death. GPD2 is highly expressed in brown adipose tissue and plays a significant role in energy production and thermogenesis. It is regulated by multiple factors, including transcriptional and post-translational mechanisms. GPD2 has been linked to diabetes, muscle regeneration, and ischemic diseases. Overexpression of GPD2 can enhance glucose oxidation and support acetyl-CoA production, which may contribute to cancer growth. In cancer, GPD1 generally acts as a tumor suppressor, while GPD2 acts as a tumor promoter. GPD1 is often downregulated in cancer tissues, and its overexpression can inhibit cancer cell growth. GPD2 is upregulated in many cancer types and is correlated with poorer survival in some cases. GPD2 regulates cancer progression through various mechanisms, including ROS generation, bioenergetics, and ether lipid biosynthesis. Overall, the review highlights the complex roles of GPD1 and GPD2 in metabolic diseases and cancer, emphasizing the need for further research to fully understand their functions and develop effective therapeutic strategies.