Abstract Signal transducer and activator of transcription 3 (STAT3) exists in 2 alternatively spliced isoforms, STAT3α and STAT3β. Although truncated STAT3β was originally postulated to act as a dominant-negative form of STAT3α, it has been shown to have various STAT3α-independent regulatory functions. Recently, STAT3β gained attention as a powerful antitumorigenic molecule in cancer. Deregulated STAT3 signaling is often found in acute myeloid leukemia (AML); however, the role of STAT3β in AML remains elusive. Therefore, we analyzed the STAT3β/α messenger RNA (mRNA) expression ratio in AML patients, where we observed that a higher STAT3β/α mRNA ratio correlated with a favorable prognosis and increased overall survival. To gain better understanding of the function of STAT3β in AML, we engineered a transgenic mouse allowing for balanced Stat3β expression. Transgenic Stat3β expression resulted in decelerated disease progression and extended survival in PTEN- and MLL-AF9–dependent AML mouse models. Our findings further suggest that the antitumorigenic function of STAT3β depends on the tumor-intrinsic regulation of a small set of significantly up- and downregulated genes, identified via RNA sequencing. In conclusion, we demonstrate that STAT3β plays an essential tumor-suppressive role in AML.