Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver non-parenchymal cells have gained increasing attention for their potential role in the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target.