Heat stress severely limits crop yield and threatens global food security; it also significantly influences the growth and development of alfalfa. Although there have been some studies on heat stress at the organ and tissue levels, the heat stress response mechanisms on the cell type level still remain unclear in alfalfa. Here, single-nucleus RNA sequencing (snRNA-seq) was performed on 61,835 high-quality leaf cells under control and heat-stressed conditions (1 and 48 h), revealing distinct transcriptional responses across 5 major cell types identified from 17 cell clusters. A total of 5,098 differentially expressed genes (DEGs) were detected, and cell type–specific regulators involving lignin biosynthesis (PAL, 4CL, OMT, CCoAOMT), hormone signaling (Aux, IAA, ARP, PIN, ERF, EIN, ASR), and kinase cascades (LRR-RLK, PP2C, WAK, and STPK) in vascular, epidermal, and mesophyll cells, respectively, were uncovered by pseudo-time trajectory analyses. The weighted gene co-expression network analysis (WGCNA) identified 10 cell type–specific heat-responsive networks, and some key heat-responsive genes (HSF, HSP, MAPK, CML, CIPK, and WRKY) exhibited opposing expression patterns in different cell types, highlighting the complexity regulatory mechanisms across cell types under heat stress. Furthermore, whole-genome bisulfite sequencing (WGBS) identified 6,252 differentially methylated regions and revealed a global increase in DNA methylation under heat stress. By integrating snRNA-seq, WGBS, and pseudo-time trajectory analyses, the core transcription factors MsHSP90 and MsHSFA5 were found to appear repeatedly. Functional studies demonstrated that MsHSP90 and MsHSFA5 significantly influence plant thermotolerance. Overall, our study provided valuable insights into the heat stress response mechanisms of alfalfa at the single-cell level.