B-cell activation occurs via antigen binding to its membrane immunoglobulin along with T-cell help to initiate intracellular signaling cascades. B cell activation enhances glucose uptake, the phosphoinositide-3-kinase (PI3K) pathway, and glycolysis and oxidative phosphorylation in a balanced manner relative to resting B-cells. The enzyme Hexokinase (HK) is central to B cell activation as it catalyzes the first step of glucose metabolism by phosphorylating glucose to become glucose-6-phosphate, which then serves as an entry point for several metabolic pathways. Hexokinase-II (HKII) is one of four HK isoforms and is particularly interesting as the only isoform that can translocate between the cytoplasm and mitochondria. It is unknown if HKII, in comparison to the other isoforms, preferentially guides glucose-6-P down a particular metabolic pathway which may depend on the B-cell’s activation status and HKII’s cellular localization. B-cell activation through the T-cell dependent signals and the PI3K pathway reprograms resting B-cell metabolism by the modification of HKII expression and subcellular localization to meet the B-cell’s new activated metabolic program. B-cells with unusual HKII expression or subcellular localization may be a feature of B-cell metabolic dysregulation, such as that observed in Chronic Lymphocytic Leukemia (CLL). A novel intracellular staining and flow cytometry assay was developed to measure HKII expression levels across B-cell subsets throughout the body and a fluorescence microscopy based assay quantified HKII mitochondrial localization. A biological mechanism is presented herein, where B-cell activation dependent on the PI3K pathway significantly increased HKII expression and along with the presence of glucose enhanced HKII mitochondrial localization. CLL B-cells failed to increase HKII expression following in vitro B-cell activation and expressed significantly less HKII, which decreased with advanced clinical Rai stage, compared to control B-cells; suggesting CLL B-cells may decrease HKII expression beyond normal levels as an anergic metabolic response to limit chronic B-cell activation. A B-cell specific HKII knockout mouse model assessed the impact of HKII deletion on B-cell subset frequencies, antibody production, and metabolism. HKII deficiency significantly reduced activated B-cells, germinal centre B-cells, and plasma cell frequencies post sheep red blood cell immunization. Plasma blasts and immunization induced plasma cells had the highest HKII expression levels across B-cell subsets and HKII played a functional role during a B-cell immune response since HKII deficiency significantly reduced antibody production in vitro and in vivo. The impact that HKII expression and localization has on B-cell metabolism during resting and activated conditions is evaluated by mass spectrometry metabolite profiling, carbon tracing using 13C labelled glucose, and a seahorse assay to quantify the ATP production rate from glycolysis and oxidative phosphorylation. HKII acted as an energy switch guiding the transition from rest to B-cell activation by fine tuning the activation of catabolic and anabolic pathways. During rest, when glucose is scare, HKII promoted glycogen breakdown for energy but facilitated the anabolic production of nucleotides and hexosamine biosynthetic pathway metabolites. During B-cell activation, when intracellular glucose is in excess, HKII promoted glycogen synthesis for energy storage and played a catabolic role in elevating the glycolytic and total ATP production rate and citric acid cycle metabolite levels. HKII is a functionally important component of B cell metabolic reprogramming dependent on the PI3K pathway and may act as an energy sensor to guide the transition from B-cell rest to activation that coincides with a switch from glycogen breakdown to synthesis depending on glucose availability.