The density of Iba1-positive cells related to microglial activation increased in the ipsilateral L4–L6 spinal dorsal horn in response to osteosarcoma development compared to the respective sham-operated groups on day 14 both in male (male: 8548.35 cell/mm3, female: 7390.88 cell/mm3) and female mice, but not on day 28, with no significant differences between the two sexes. Here, AIF1 is linked to osteosarcoma.