The active vitamin D metabolites, 25-hydroxyvitamin D$_3$ (25D$_3$) and 1,25-dihydroxyvitamin D$_3$ (1,25D$_3$), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D$_3$ is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D$_3$. Structural analysis indicates that 1,4α,25(OH)$_3$D$_3$ and 1,4β,25(OH)$_3$D$_3$ maintain the anchoring hydrogen bonds of 1,25D$_3$ and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D$_3$ and 1,4β,25D$_3$ are as potent as 1,25D$_3$ in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.