The present study was undertaken to determine the involvement of voltage-gated Na⁺ channel (VGSC) and other mechanism related to insulin secretion in polysaccharides from Portulaca oleracea L. (POP)-induced secretion of insulin from insulin-secreting β-cell line cells (INS-1) cells. Our results showed that the concentration of insulin both in culture medium and inside INS-1 cells were increased under the existing of different concentration of glucose by POP or TTX, respectively. However, the effect POP on insulin secretion and production were blocked by TTX, a VGSC blocker. Meanwhile, POP improved the mitochondrial membrane potential (Δψm), increased adenosine triphosphate (ATP) production, depolarized cell membrane potential (MP) and increased intracellular Ca²⁺ levels ([Ca²⁺]_i). Furthermore, POP treatment increased the expression level of Nav_1.3 and decreased the expression level of Nav_1.7. TTX treatment decreased the expression level of Nav_1.3 and Nav_1.7. On the other hand, POP also elevated the survival of INS-1 cells. These results suggested that POP induced-secretion/production of insulin in INS-1 cells were mediated by VGSC through its change of function and subunits expression and subsequent VGSC- dependent events such as change of intracellular Ca²⁺ releasing, ATP metabolism, cell membrane and mitochondrial membrane potential, and also improvement of INS-1 cell survival. Meanwhile, our data indicated the potentiality of developing POP to be a drug for diabetes treatment and VGSC as a therapeutic target in diabetes treatment is valuable to be investigated further.