Lipid metabolism dysregulation is a critical factor contributing to obesity. To counteract obesity-associated disorders, bariatric surgery is implemented as a very effective method. However, surgery such as Roux-en-Y gastric bypass (RYGB) is irreversible, resulting in life-long changes to the digestive tract. The aim of the present study was to elucidate changes in the fecal microbiota before and after RYGB in relation to blood lipid profiles and proinflammatory IL-6. Here, we studied the long-term effects, up to six years after the RYGB procedure, on 15 patients’ gut microbiomes and their post-surgery well-being, emphasizing the biological sex of the patients. The results showed improved health among the patients after surgery, which coincided with weight loss and improved lipid metabolism. Health changes were associated with decreased inflammation and significant alterations in the gut microbiome after surgery that differed between females and males. The Actinobacteriota phylum decreased in females and increased in males. Overall increases in the genera Prevotella, Paraprevotella, Gemella, Streptococcus, and Veillonella_A, and decreases in Bacteroides_H, Anaerostipes, Lachnoclostridium_B, Hydrogeniiclostridium, Lawsonibacter, Paludicola, and Rothia were observed. In conclusion, our findings indicate that there were long-term changes in the gut microbiota after RYGB, and shifts in the microbial taxa appeared to differ depending on sex, which should be investigated further in a larger cohort.