In this study, a new integrative vector (pBCGA) was developed and used for the high-level expression of an optimized iota-carrageenase gene (CGIOP) in Brevibacillus choshinensis. The pBCGA vector allowed multiple copies of the gene CGIOP to be stably integrated into the genomic DNA of B. choshinensis. The recombinant strain 124 could produce an extracellular iota-carrageenase activity of 38.9 U/mL within 72 h, which remained stable after five sequential inoculations and cultivations under the antibiotic-free culture conditions. Furthermore, the strain 124 was applied to 10-L fermentation under the antibiotic-free culture condition, resulting in the highest observed iota-carrageenase activity of 182.4 U/mL within 24 h. Subsequently, recombinant iota-carrageenase (rCgiA) was purified and characterized, exhibiting an optimal pH and temperature of 8.0 and 45 degrees C, respectively. Notably, rCgiA showed considerable stability below 45 degrees C and over a relatively broad pH range of 6.0-11.0. In addtion, the activity of rCgiA was significantly stimulated by NaCl, Mg2+, and Ca2+. HILIC LC-LTQ-Orbitrap-FTMS analysis revealed that rCgiA hydrolyzed iota-carrageenan via a processive mechanism with the major product of iota-carrageenan tetrasaccharide. Thus, the strain B. choshinensis 124 had broad potential for use in the environment-friendly and large-scale production of iota-carrageenase and iota-carrageenan oligosaccharides.