Plasmid-freeLactococcus lactisIL1403 is one of the best-characterized representatives of lactic acid bacteria (LAB), intensively used in broad microbiology worldwide. Its parent strain,L. lactisIL594, contains seven plasmids (pIL1–pIL7) with resolved DNA sequences and an indicated role for overall plasmid load in enhancing host-adaptive potential. To determine how individual plasmids manipulate the expression of phenotypes and chromosomal genes, we conducted global comparative phenotypic analyses combined with transcriptomic studies in plasmid-freeL. lactisIL1403, multiplasmidL. lactisIL594, and its single-plasmid derivatives. The presence of pIL2, pIL4, and pIL5 led to the most pronounced phenotypic differences in the metabolism of several carbon sources, including some β-glycosides and organic acids. The pIL5 plasmid also contributed to increased tolerance to some antimicrobial compounds and heavy metal ions, especially those in the toxic cation group. Comparative transcriptomics showed significant variation in the expression levels of up to 189 chromosomal genes due to the presence of single plasmids and 435 unique chromosomal genes that were resultant of the activity of all plasmids, which may suggest that the observed phenotypic changes are not only the result of a direct action of their own genes but also originate from indirect actions through crosstalk between plasmids and the chromosome. The data obtained here indicate that plasmid maintenance leads to the development of important mechanisms of global gene regulation that provide changes in the central metabolic pathways and adaptive properties ofL. lactisand suggest the possibility of a similar phenomenon among other groups of bacteria.