Signal transducer and activator of transcription 3 (STAT3) dramatically increases during the first post-natal week, and supports the survival of mature hippocampal neurons. Recently, we reported that chronic elevation of excitability leads to a loss of STAT3 signal, inducing vulnerability in neurons. The loss of STAT3 signal was due to impaired Erk1/2 activation. While overnight elevation of activity attenuated STAT3 signal, brief low-frequency stimuli, which induce long-term depression, have been shown to activate STAT3. Here we investigated how STAT3 responds to depolarization in mature neurons. A brief depolarization results in the transient activation of STAT3: it induces calcium influx through L-type voltage-gated calcium channels, which triggers activation of Src family kinases. Src family kinases are required for phosphorylation of STAT3 at Tyr-705 and Ser-727. PTyr-705 is Janus kinase (JAK)-dependent, while PSer-727 is dependent on Akt, the Ser/Thr kinase. Both PTyr-705 and PSer-727 are necessary for nuclear translocation of STAT3 in these neurons. Chronic elevation of spontaneous activity by an A-type potassium blocker, 4-aminopyridine (4-AP), also induced the transient phosphorylation of STAT3, which after 4 h fell to basal levels despite the presence of 4-AP. These results suggest that phasic and chronic neuronal activation induce distinct molecular pathways, resulting in opposing regulation of STAT3 signal.