Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) is widely used as a starter for yogurt and cheese worldwide. Despite the economic importance of this bacterium in the dairy industry, there have been few genetic studies involving knockout or overexpression mutants to identify the functions of Lb. bulgaricus genes. One of the main reasons for this gap is the low transformation efficiency of available Lb. bulgaricus chromosome-integrating vectors upon performing conventional electroporation. We previously proposed the conjugal plasmid pAMβ1 as an integration vector for Lb. bulgaricus, as conjugation could avert the need for a restriction 2 modification system; pAMβ1 does not replicate and integrate into the chromosome of Lb. bulgaricus. Here, we describe an effective chromosomal manipulation system involving a novel shuttle vector pGMβ1, which could improve the operability of the broad host-range conjugal plasmid pAMβ1. We further developed an enhanced filter-mating method for conjugation. To validate this system, the effectiveness of conversion of the lactate dehydrogenase gene D-ldh of Lb. bulgaricus to the L-ldh form of Streptococcus thermophilus was examined. As pGMβ1 and pAMβ1 are unable to replicate in Lb. delbrueckii subsp. delbrueckii, they were chromosomally integrated.However, these plasmids could replicate in Lb. delbrueckii subsp. indicus and sunkii.This integration system could unearth important gene functions in Lb. bulgaricus and thus improve its applications in the dairy industry. Moreover, this conjugation system could be used as a stable vector for the transformation of long cluster genes in several species of lactic acid bacteria.