White adipose tissue (WAT) has a critical role in lipid handling. Previous work demonstrated that SCD1 is an important regulator of WAT fatty acid (FA) composition; however, its influence on the various interconnected pathways influencing WAT lipid handling remains unclear. Our objective was to investigate the role of SCD1 on WAT lipid handling using Scd1 knock-out (KO) mice and SCD1-inhibited 3T3-L1 adipocytes by measuring gene, protein, and metabolite markers related to FA re-esterification, glyceroneogenesis, and lipolysis. Triacylglycerol (TAG) content was higher in inguinal WAT (iWAT) from KO mice compared to wild-type (WT), but significantly lower in epididymal WAT (eWAT). The SCD1 desaturation index was decreased in both WAT depots in KO mice. FA re-esterification, as measured with a NEFA:glycerol ratio, was reduced in both WAT depots in KO mice, as well as SCD1-inhibited 3T3-L1 adipocytes. Pck1, Atgl, and Hsl gene expression were reduced in both WAT depots of KO mice, while Pck2 and Pdk4 gene expression showed depot-specific regulation. Pck1, Atgl, and Hsl gene expression were reduced, and PEPCK protein content ablated, in SCD1-inhibited adipocytes. Our data provides evidence that SCD1 has a broad impact on WAT lipid handling by altering TAG composition in a depot-specific manner, reducing FA re-esterification, and regulating markers of lipolysis and glyceroneogenesis.