Objective To investigate the effect of estrogen deficiency on the degeneration of spinal endplate chondrocytes. Methods Forty 6-month-old rats were divided into ovariectomy (OVX) and sham operation groups. All of the rats were sacrificed at week 9 after operation. The cartilage endplate tissue was extracted, and endplate chondrocytes were cultured. The expression of COL-II in the cartilage endplate tissue was evaluated by immunohistochemistry. The cells were stained by toluidine blue to identify endplate chondrocytes. The viability of endplate chondrocytes was detected by CCK-8. Fluorescence staining of rhodamine-labeled phalloidin was used to observe the changes of F-actin after OVX. Cellular immunofluorescence was performed to detect the changes of COL-II of endplate chondrocytes. The expression levels of SOX9, ACAN, ADAMTS-5, MMP13 and COL-X were compared between the two groups by RT-qPCR. Results After OVX, the expression of COL-II protein in the cartilage endplates was decreased. Most of the chondrocytes in the endplates were polygonal and fusiform, arranged like paving stones. Endplate chondrocytes of OVX demonstrated more disordered cytoskeleton, decreased viability, increased stress fibers, decreased migration ability, and lower COL-II expression. For the OVX group, the expression of SOX9 and ACAN in the endplate chondrocytes was decreased, while that of ADAMTS-5, MMP13 and COL-X was increased. Conclusions Estrogen deficiency can cause degeneration of endplate chondrocytes.