Due to the importance of the gut microbiota in the regulation of energy homeostasis, probiotics have emerged as an alternative therapy to ameliorate obesity-related disturbances, including cholesterol metabolism dysregulation, dyslipidemia and inflammation. Therefore, the objectives of this study were to evaluate the effect of the probiotic strain Pediococcus acidilactici (pA1c®) on the regulation of adiposity, cholesterol and lipid metabolism, inflammatory markers and gut microbiota composition in diet-induced obese rats. Twenty-nine four-week-old male Wistar rats were divided into three groups: rats fed a control diet (CNT group, n = 8), rats fed a high fat/high sucrose diet (HFS group, n = 11), and rats fed a HFS diet supplemented with pA1c® (pA1c®group, n = 10). Organs and fat depots were weighed, and different biochemical parameters were analysed in serum. Gene expression analyses in the adipose tissue were conducted using real-time quantitative-PCR. Faecal microbiota composition was evaluated using 16S metagenomics. Animals supplemented with pA1c® exhibited a lower proportion of visceral adiposity, a higher proportion of muscle, an improvement in the total-cholesterol/HDL-cholesterol ratio and a decrease in the total cholesterol, triglyceride and aspartate aminotransaminase (AST) serum levels, together with a decrease in several inflammation-related molecules. The expression of key genes related to adipose (Adipoq, Cebpa and Pparg) and glucose (Slc2a1 and Slc2a4) metabolism in the adipose tissue was normalized by pA1c®. Moreover, it was demonstrated that pA1c® supplementation activated fatty acid β-oxidation in the adipose tissue and the liver. Metagenomics demonstrated the presence of pA1c® in the faecal samples, an increase in alpha diversity, an increase in the abundance of beneficial bacteria, and a decrease in the abundance of harmful micro-organisms, including the Streptococcus genus. Thus, our data suggest the potential of pA1c® in the prevention of obesity-related disturbances including hypercholesterolemia, hypertriglyceridemia, inflammation and gut microbiota dysbiosis.