The objective of the study was to describe the feasibility of a glove port technique for laparoscopic-assisted surgical treatment of canine pyometra. In this retrospective case series, a total of 10 female dogs (median age 7 years, range 5.5-10.5 years; median weight 37.0 kg, range 12.9-64.0 kg) with pyometra were included. A multiaccess port was created from a surgical glove attached to an Alexis wound retractor and placed in the ventral midline between the middle and caudal third of the distance between umbilicus and pubic rim. A vessel sealing device was used for transection of the ovarian pedicle. The port size was selected on the basis of maximum uterine diameter determined by ultrasound. Median incision length was 5.0 cm (range 3.1-7.7 cm) for a maximum uterine diameter of 4.0 cm (range 2.0-7.0 cm). Median surgical time was 57 minutes (range 48-65 minutes). No case had to be converted to open celiotomy. Complications included one case of minor, self-limiting splenic trauma by the endoscope. In eight dogs, the distended uterine horns endangered safe access to the ovarian pedicle, and the vessel sealing device was inserted through a second cannula placed periumbilically. Extension of the original incision was necessary to exteriorize organs in two dogs. All dogs recovered quickly and were discharged either on the day of surgery or 1 day thereafter. In conclusion, a surgical glove port technique in combination with an Alexis wound retractor is feasible for surgical laparoscopic treatment of canine pyometra up to a diameter of 7 cm.