5,6-trans-1alpha,25-Dihydroxyvitamin D3 is a synthetic analog of the active form of vitamin D, calcitriol (1α,25-dihydroxyvitamin D3). It features a modified stereochemistry at the C-5 and C-6 positions of the seco-steroid B-ring, resulting in the 5,6-trans configuration. This structural alteration distinguishes it from the natural hormone. This compound is a potent agonist of the vitamin D receptor (VDR). It exhibits biological activities similar to calcitriol, including the regulation of calcium and phosphate homeostasis, cell differentiation, and immune modulation. Due to its activity, it has been extensively used as a research tool in biochemical and pharmacological studies to investigate the mechanisms of vitamin D action, its receptor signaling pathways, and its role in various physiological and pathological processes, such as bone metabolism, cancer, and autoimmune diseases. As a research-grade biochemical, it is not used as a therapeutic API in clinical practice but serves as a critical reference standard and probe in life science research. Its synthesis and availability support ongoing investigations into vitamin D endocrinology and the development of novel vitamin D analogs with potential therapeutic applications.