Background: Tillandsia usneoides (Bromeliaceous) is traditionally used in Mexico for diabetes treatment. Although a significant hypoglycemic effect has been reported, the participation of insulin in this action has not yet been explored. Objectives: The aim of this research was to determine the hypoglycemic effect of an aqueous extract from T. usneoides in normal and diabetic mice as well as to evaluate the participation of insulin in this effect using an in vitro model. Materials and Methods: Aqueous decoction of T. usneoides (250 mg/kg) was administered in healthy and diabetic mice and glycemia was measured. RINm5F cells were cultured with T. usneoides (0.1 and 1 μg/ml), and cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, trypan blue, and apoptosis; secretion and expression of insulin were quantified by enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction, respectively. Results: T. usneoides decreased blood glucose in healthy mice at 4 and 6 h (96 ± 11.2 and 68.2 ± 1.9 mg/dl, respectively) compared with time zero (138.5 ± 5.0 mg/dl, P < 0.05). In diabetic mice, aqueous extract significantly decreased glycemia at 6 h compared with time zero (212.7 ± 3.5 and 243 ± 5.3 and mg/dl, respectively). Besides, T. usneoides aqueous extract stimulated insulin secretion (20%, P < 0.05) without cause changes in insulin gene expression and protects RINm5F cells from streptozotocin-induced apoptosis. Conclusion: These results suggest that aqueous decoction of T. usneoides stimulates insulin secretion still in the absence of changes in the intracellular concentration of Ca2+ in RINm5F cells.