The pericentric inversion of chromosome 16, inv(16)(p13q22), is associated with acute myeloid leukemia (AML) subtype M4Eo that is characterized by the presence of myelomonocytic blasts and atypical eosinophils. This rearrangement fuses the CBFB and MYH11 genes, with the latter encoding the smooth muscle myosin heavy chain (SMMHC). The myeloid transcription factor CCAAT/enhancer-binding protein alpha (CEBPA) is crucial for normal granulopoiesis. Alterations of structure and expression of CEBPA have been implicated in particular subtypes of AML. Here, we found that conditional expression of core-binding factor beta (CBFB)-SMMHC in U937 cells suppresses CEBPA protein expression and binding activity. However, CEBPA mRNA levels remained unchanged. No differences were detected in CEBPA mRNA levels in patients with inv(16) AML-M4Eo (n = 12) compared to patients with AML with a normal karyotype and M4 subtype (n = 6), whereas CEBPA protein and binding activity were significantly reduced in patients with CBFB-SMMHC. Furthermore, calreticulin, an inhibitor of CEBPA translation, was induced on mRNA and protein level in CBFB-SMMHC patients with AML and after expression of CBFB-SMMHC in the U937-cell system. Inhibition of calreticulin by siRNA restored CEBPA levels. Our results suggest that modulation of CEBPA by calreticulin represents a novel mechanism involved in the differentiation block in CBFB-SMMHC AML.