Background. Pneumoperitoneum insufflation with warmed and humidified carbon dioxide (WH-CO2) can prevent heat loss and increase tissue oxygenation. We evaluated the impact of localized WH-CO2 insufflation on the anastomotic healing process. Methods. Sixty male Wistar rats were randomized: Group 1 (control, n = 12), Group 2 (cold and dry CO2, CD-CO2, n = 24), and Group 3 (WH-CO2, n = 24). A magnetic compression side-to-side colonic anastomosis was performed under 60-minute local abdominal CO2 flow insufflation. Animal temperature was recorded. IL-1, IL-6, and CRP levels were assessed before and after insufflation and on postoperative day (POD) 7 and POD 10. Endoscopic follow-up was performed on POD 7 and POD 10. A burst pressure (BP) test of the specimen was performed on POD 10, and histopathological analysis was then performed. Metabolomics of the anastomotic site was determined. Results. Seven rats (5 CD-CO2 group, 1 WH-CO2 group, and 1 control group) died during the survival period. Necropsies revealed intestinal occlusions (n = 2). One additional rat from the CD-CO2 group was sacrificed on POD 7 due to intestinal perforation. The postoperative course was uneventful in the remaining cases. There was no difference in BP among the groups. Thermal monitoring confirmed that WH-CO2 insufflation was effective to reduce heat loss. IL-1 levels were statistically and significantly lower on POD 10 in the WH-CO2 group than the CD-CO2 group but not lower than the control group. CRP levels, histopathology, and metabolomics did not show any difference between the 3 groups. Conclusions. WH-CO2 was effective to preserve core temperature. However, it did not improve anastomotic healing.