The status of probiotics is increasing worldwide and the range of application of probiotics is also being extended to the livestock industry. The use of probiotics in the swine industry aims to improve animal health and productivity by forming a healthy gut microbiota. Intensive pig breeding, a common breeding method in modern society, causes physiological stress in pigs, resulting in imbalance of the intestinal microbiota and dysfunction of the intestinal barrier. We conducted a scientific research on the properties of probiotics for multi-strain probiotics (MSP) and observed the economic benefits and efficiency of culturing MSP through a self-cultivation system in livestock. Initially, we observed alteration occurred by MSP application in the gut microbiota of pig. MSP were characterized to have resistance to digestive juices such as gastric acid and bile, followed by colonization in the target organ, the gastro-intestinal tract (GIT). In addition, MSP was also confirmed to have antibacterial ability against pathogenicity that most frequently infected in the swine industry and showed low resistance to antibiotics which means guaranteed stability when added to feed. The growth rate of probiotics in the optimized medium used in the self-cultivation system was suitable for efficiently and economically culturing and feeding pigs with high concentrations of probiotics, considering the ingredient price and the growth efficiency. We observed the significant alteration of gut microbiota of pig by application of MSP. Importantly, the MSP supplementation significantly increased the beneficial bacteria genus of Bifidobacterium that confer a health benefit to the host in pig GIT, whereas decreased the number of harmful bacteria including coliforms. In addition, MSP influenced on the uniformity of gut microbiota at both of sow and weaning pigs. Taken together, the application of MSP with high concentration of probiotics using self-cultivation system may critically improve the pig health by regulation intestinal microbiota.