T cells engineered with chimeric antigen receptor (CAR-T) have shown success in treating hematological malignancies. However, the limited efficacy and potential side effects hinder the use of CAR-T in solid tumors. Research on this heterogeneous cell therapeutic at the single-cell level can advance the current understanding and strategy of CAR-T therapy. We used single-cell transcriptome analysis to characterize the transcriptional profile of stimulated CAR-T. T cells engineered with second-generation CAR constructs (with CD28 or 4-1BB co-stimulatory domains) were stimulated with anti-CD3/CD28-coated beads and Nalm6 cells and subjected to microwell-based single-cell transcriptome analysis. A broad spectrum of transcripts was differentially expressed in T cells under various conditions. The sequence derived from the 3’-untranslated region of the CAR transcript distinguished CAR-T from non-transduced T cells within the analyzed cell population. CAR-T stimulation, mediated by either CD3/CD28 or CAR, significantly altered the gene expression landscape compared to unstimulated CAR-Ts. Notably, CD3/CD28 stimulation triggered upregulation of T cell signaling-related genes compared to CAR stimulation. Additionally, comparing CD28 and 4-1BB co-stimulatory domains showed distinct transcriptional profiles, including cell division, chemotaxis, and cell killing. This study revealed characteristic features of CAR-T signals affected by receptor structure (CD3/CD28 vs. CAR) and co-stimulatory domain (CD28 vs. 4-1BB). These findings provide insights into a deeper understanding of CAR-T therapy, ultimately leading to the development of more effective CAR-T therapy.