Stromal interaction molecule (STIM1) is the main regulator for basal cytoplasmic calcium levels via store-operated calcium entry (SOCE). SOCE dysregulation has been reported to be involved in numerous cancers development and metastasis. However, the association of STIM1 and SOCE in acute myeloid leukemia (AML) pathogenesis remains unclear. The present work investigates the SOCE activities via Orai1 expression and cytosolic free calcium level assessment in STIM1 silencing AML cells such as THP-1 and Kasumi-1 cells. STIM1 and Orai1 expressions were profiled through real-time reverse transcription PCR (qRT-PCR) platform. The changes in the cytosolic free calcium levels were quantified using the intracellular calcium indicator fura-2 acetoxymethyl ester (Fura-2AM). This study revealed that STIM1 and Orai1 were expressed in both AML cell lines. STIM1 expression was observed more than 2.5-fold higher in THP-1 cells compared to Kasumi-1 cells, which could be related to the aggressive phenotype of THP-1 cells with M5 subtype rather than Kasumi-1 cells with M2 subtype. STIM1 knockdown has shown a regularity effect on SOCE activities by suppression of Orai1 and decreases the calcium influx profile, especially in THP-1 cells. This observation supports the importance of STIM1 in the regulation of calcium homeostasis in AML through SOCE. Findings from this study suggest that STIM1 may serve as a potential therapeutic target for AML, especially for aggressive FAB subtypes. However, further comprehensive work is needed to support these findings.