Heat stress and extreme temperatures negatively affect plant development by disrupting regular cellular and biochemical functions, ultimately leading to reduced crop production. Recently, our group has shown through physiological experiments that miR156 overexpression resulted in an improved alfalfa response to heat stress. To further expand the scope of miR156 research, we employed a label-free quantification based quantitative proteomics approach to explore the effects of heat stress on protein levels in miR156OE alfalfa. Our major objective was to identify miR156-regulated gene products with differentially altered abundance under heat stress in alfalfa.