Signal transducer and activator of transcription (STAT) proteins are activated by phosphorylation in the spinal cord of patients suffering from amyotrophic lateral sclerosis (ALS). The major scope of our study is a comprehensive histological characterization of the mechanisms underlying neuronal and glial STAT3 activation in the pathogenesis of ALS using SOD1(G93A) mice. We calculated the fold changes (FCs, ratios vs. appropriate controls) of the numerical densities of the following phosphorylated STAT3-positive (pSTAT3)(+) cells - choline acetyltransferase (ChAT)(+) α-motoneurons, ionized calcium-binding adapter molecule 1 (Iba1)(+) microglia, and S100β(+) astrocytes in SOD1(G93A) mice. The FCs of pSTAT3(+) microglia and pSTAT3(+) astrocytes were increased from 9 to 15 weeks of age and then plateaued until 21 weeks. In contrast, the FCs of pSTAT3(+) α-motoneurons peaked at 9 weeks and then decreased until 21 weeks. The immunoreactivity for nonphosphorylated neurofilament protein (SMI-32), a marker of axonal impairment, was decreased in pSTAT3(+) α-motoneurons compared with pSTAT3(-) α-motoneurons at 9 weeks of age. We then compared the following pharmacological models - the chronic administration of 3,3'-iminodipropionitrile (IDPN), which models axonal impairment, and the acute administration of lipopolysaccharide (LPS), which is a model of neuroinflammation. The FCs of pSTAT3(+) α-motoneurons were increased in IDPN-treated mice, while those of pSTAT3(+) microglia were increased in LPS-treated mice. The FCs of pSTAT3(+) astrocytes were higher in SOD1(G93A) mice at 9 weeks compared with IDPN- and LPS-treated mice. Our results indicate that axonopathy and neuroinflammation may trigger the respective activation of neuronal and glial STAT3, which is observed during ALS pathogenesis.