We herein adapted a markerless gene replacement method by combining a temperature-sensitive plasmid pKSV7 with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the poly-gamma-glutamic acid (gamma-PGA)-producing strain Bacillus amyloliquefaciens LL3. Deletion of the upp gene conferred LL3 5-fluorouracil (5-FU) resistance. Sensitivity to 5-FU was restored when LL3 Delta upp was transformed with pKSV7-based deletion plasmid which carries a functional allele of the upp gene of Bacillus subtilis 168. These observations allowed us to adapt a two-step plasmid integration and excision strategy to perform markerless deletion of genes of interest. Deletion plasmid harboring a mutant allele of the target gene was first integrated in the genome by culturing cells under nonpermissive conditions for pKSV7 replication. Single-crossover recombinants were then grown without antibiotics to aid the second recombinational event. 5-FU was used to select for double-crossover recombinants with plasmid evicted from the chromosome. The resulting recombinants either harbored the wild-type or mutated allele of the target gene and could be identified by PCR and DNA sequencing. Using this method, we successively removed the amyA gene and a 47-kb fragment of the bae cluster from the genome of LL3, with higher efficiency compared with previous reports. We also investigated the effects of a transcriptional regulator, RocR, on gamma-PGA production and cell growth. Specific gamma-PGA production of the rocR mutant was increased by 1.9-fold, which represents a new way to improve gamma-PGA production.