Lactococcus is an economically important group in lactic acid bacteria (LAB) that are often used in the dairy industry as starters for cheese production. Good starter strains should possess the ability to grow, ferment milk sugar, and produce desirable flavor compounds during cheese making. Therefore, it is essential to understand the physiology of these starters during cheese processing in order to obtain high-quality cheese products. Cheese manufacturing compromises several stress factors that affect the growth of starter lactococci. Among these stressed environmental parameters, sugar starvation is the most important one to overcome to obtain energy for cellular processes. It is known that degradation of arginine produces energy. In this study, we investigated arginine utilization by Lactococcus lactis ssp. lactis strain ML3 via the arginine deiminase (ADI) pathway to see its influence on cellular physiology after exhaustion of a primary energy source. During the cell growth in a carbohydrate-limited environment, we observed that metabolic pathways switched between lactose utilization arginine degradation. The statistical model described in this study suggested lactose and arginine were co-metabolized during cell growth. These results initially showed arginine was a good candidate of secondary energy source after exhaustion of primary energy source (lactose). To confirm these observations, cell counts, cellular ATP levels, ADI enzyme activities, and total protein expression were compared in arginine-positive L. lactis ssp. lactis ML3 and arginine-negative L. lactis ssp. cremoris Sl grown in medium containing 0.2% lactose and 2% arginine. Results showed ATP levels remained high in strain ML3, in which a transition stage of protein expression pattern was also observed. This physiological evidence highlights the important roles of arginine degradation in starved ML3, perhaps by producing extra ATP and modulating external pH. The genes involved in the ADI pathway of strain ML3 were cloned, sequenced, and characterized. Genes involved in this pathway formed a unique multi-operon cluster structure that we termed MOC. It was organized as arcA, arcBD1, arcC1C2, and arcTD2. The influence of different environmental parameters including pH, various amino acids, and phosphate (organic and inorganic) on the expression of the ADI MOC was tested. No single factor regulated the entire MOC simultaneously. It is concluded that the unique structure of the MOC appears to allow the ADI pathway to occur in discrete sections in response to fluctuated external conditions, such as sugar starvation and low environmental pH.