Background <p>The cell fate transition of pluripotent callus from somatic cells plays an important role in plant development studies and crop genetic improvement. Embryonic callus is a group of totipotent cells derived from the founder cells responsible for the initiation of callus. However, our understanding of the initial cellular states and regulatory factors of cell fate transition during callus development remains quite limited.</p> Results <p>Single-cell RNA sequencing was employed to generate a single-cell transcriptome atlas of the rice (<i>Oryza sativa</i> L.) embryonic and differentiated callus. We found that the callus founder cell represented the transcriptionally earliest state of embryonic callus, which could transit into the root meristem niche, a process associated with starch degradation, lipid transport, energy production and transfer, plant hormones and flavonoid metabolism. The shoot meristematic cell represented the transcriptionally earliest state of differentiated callus, and these shoot meristematic cells differentiated into mesophyll precursor cells via vascular parenchyma cells, a process associated with chromatin-related epigenetic regulation, enzymatic reactions, signal transduction, metabolic activity and photosynthesis. The expression patterns of the genes involved in plant hormones, and <i>WUSCHEL RELATED HOMEOBOX</i> (<i>WOX</i>) family gene <i>DWARF TILLER1</i> (<i>OsDWT1</i>) across distinct cell populations suggested that gibberellin (GA) signaling and <i>OsDWT1</i> were associated with the callus founder cell fate transition.</p> Conclusions <p>Our results revealed that the callus founder cell and shoot meristematic cell represent the transcriptionally earliest states of embryonic callus and differentiated callus in rice, respectively. This study provides preliminary resources for understanding the development of callus cells and offers clues for improving plant genetic transformation efficiency in recalcitrant rice varieties by regulating cell fate transition.</p>