Plants experienced modest changes in ambient temperature, which affects primarily their flowering time, in most of their life cycle. The microRNA156-SPL3 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3) module directly regulates FT expression in the leaf to modulate plants’ flowering response to ambient temperature changes. Overexpression of miR156 caused increased sensitivity to ambient temperature changes. Anti-correlated pattern between mRNA levels and cleavage products of SPL3 at low temperature suggested that SPL3 plays a major role in miR156 function. Genetic interaction studies suggested that FT is a major output of the miR156-SPL3 module in ambient temperature signaling and that FUL acts downstream of FT. SPL3 protein directly binds to the consensus GTAC motifs (-219 to -176) of the proximal region of the FT promoter. It was suggested that regulation of miR156-SPL-FT module is part of regulation mechanism of flowering time in response to ambient temperature changes. In order to understand, the correlation between the biogenesis of primary miRNA156a and flowering delay induced by miR156-SPL3 module, we investigated the kinetics and thermodynamics for base-pair opening of the model pri-miRNA156a by using NMR spectroscopy. Comparison of the wild-type and mutants pri-miRNA156a indicates that some clear differences in base-pair opening properties are correlated with relative efficiency of maturation of pri-miRNA156a by DCL1 proteins.