The development of rBAY, a recombinant peptide with the similar sequence of synthetic BAY55-9837, as a potential peptide therapeutic for type 2 diabetes is still a challenge mainly because of its poor stability in aqueous solution. To improve the peptide stability and bioactivity and investigate its biological effects for VPAC2-specific activation, RBAYL with 31 aa was designed based on sequence alignments of pituitary adenylate cyclase-activating peptides (PACAPs), vasoactive intestinal peptide (VIP), and related analogs and generated through sitedirected mutagenesis. Stability analysis showed that the prepared RBAYL with three mutations (N9Q, V17L, and N28K) were much more stable than rBAY. rRBAYL (the recombinant RBAYL) was expressed and purified by gene-recombination technology via native thiol ligation on solid beads. As much as 27.7 mg rRBAYL peptide with purity over 98% was obtained from 1 L of LB medium without expensive high-performance liquid chromatography refinements. The bioactivity assay of rRBAYL showed that it displaced [(125)I]PACAP38 and [(125)I]VIP from VPAC2 with a half-maximal inhibitory concentration of 51+/-6 and 50+/-4 nM, respectively, which were similar to those of the chemically synthesized RBAYL (sRBAYL) and lower than those of Ro25-1553, an established VPAC2 agonist. rRBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC50) of 0.91 nM, whereas the receptor potency of rRBAYL at human VPAC1 (EC50 of 719 nM) was only 1/790 of that at human VPAC2, and rRBAYL had no activity toward human PAC1 receptor. Western-blot assay for glucose transporter 4 (GLUT4) indicated that the rRBAYL could significantly induce GLUT4 expression more efficiently than rBAY or Ro25-1553 in adipocytes. Compared with rBAY, rRBAYL can more efficiently promote insulin release and decrease plasma glucose level in ICR mice. Our results suggested that rRBAYL is a novel recombinant VPAC2-specific agonist with high stability and bioactivity.