Florida manatees (Trichechus manatus latirostris) continue to experience pressure from various stressors that frequently result in the need for rescue and veterinary assistance. Interestingly, a subset of rescued manatees in critical condition exhibits profound hypoglycemia. The goals of this study were to enhance our understanding of this important aspect of manatee care by 1) characterizing the clinical presentation and factors associated with manatees that present with profound hypoglycemia, and 2) assessing agreement across blood glucose testing modalities [glucometer (whole blood), in-house bench-top analyzer (whole blood), a point of care analyzer (whole blood), and a local human hospital laboratory analyzer (serum)]. Twenty-five manatees were admitted to the Jacksonville Zoo and Gardens from 2017 to 2021. Seven manatees were profoundly hypoglycemic on presentation with blood glucose concentrations of < 30 mg/dL. Hypoglycemic manatees were admitted due to cold stress syndrome (n = 3; 1 calf, 2 adults) or emaciation consistent with an unusual mortality event (n = 4; 2 calves, 2 adults). Physical examination findings in hypoglycemic animals included emaciation (n = 7, 100%), lethargy (n = 7, 100%), and intermittent mild muscle fasciculations of the muzzle (n = 3, 42.8%). Hypoglycemia resolved with intense supportive care and monitoring in all affected animals within 32 h of admission. No associations were identified between hypoglycemia on presentation and life-stage, sex, transport distance to JZG's critical care facility, or rehabilitation outcome (death/euthanasia vs. release). Statistical agreement was observed between all blood glucose testing modalities except for the glucometer vs. the human hospital analyzer. Despite the limited sample size, these findings inform on the clinical presentation of severe hypoglycemia, the importance of close blood glucose monitoring after admission, blood glucose testing modalities, and clinical decision-making in Florida manatees admitted to critical care centers.