Signal transducer and activator of transcription 3 (STAT3) is an important oncogenic transcription factor residing in the cytoplasm in the resting cells. Upon stimulation, STAT3 is activated and translocated to the nucleus to regulate target genes. Although the canonical transcriptional function of STAT3 has been intensively studied, less is known about its cytoplasmic localization. In this study, by immunoprecipitation, microtubule cosedimentation, and immunofluorescence assays, we present the first evidence that cytoplasmic STAT3 interacts with both tubulin and microtubules. By using small-molecule inhibitor approaches, we further demonstrate that the localization of STAT3 on microtubules and its activation are independent of histone deacetylase 6 (HDAC6) activity. In addition, disruption of microtubule dynamics does not alter the activation and nuclear translocation of STAT3 in response to interleukin-6 treatment. These findings reveal that cytoplasmic STAT3 is physically associated with microtubules, whereas its activation and nuclear translocation are independent of microtubule dynamics, implicating that the association of STAT3 with microtubules might be involved in the regulation of noncanonical functions of STAT3 in the cytoplasm.