Spermatozoa acquire their fertilizing ability and forward motility during epididymal transit, suggesting the importance of the epididymis. Although the cell atlas of the epididymis was reported recently, the heterogeneity of the cells and the gene expression profile in the epididymal tube are still largely unknown. Considering single-cell RNA sequencing results, we thoroughly studied the cell composition, spatio-temporal differences in differentially expressed genes (DEGs) in epididymal segments and mitochondria throughout the epididymis with sufficient cell numbers. In total, 40,623 cells were detected and further clustered into 8 identified cell populations. Focused analyses revealed the subpopulations of principal cells, basal cells, clear/narrow cells, and halo/T cells. Notably, two subtypes of principal cells, the Prc7 and Prc8 subpopulations were enriched as stereocilia-like cells according to GO analysis. Further analysis demonstrated the spatially specific pattern of the DEGs in each cell cluster. Unexpectedly, the abundance of mitochondria and mitochondrial transcription (MT) was found to be higher in the corpus and cauda epididymis than in the caput epididymis by scRNA-seq, immunostaining, and qPCR validation. In addition, the spatio-temporal profile of the DEGs from the P42 and P56 epididymis, including transiting spermatozoa, was depicted. Overall, our study presented the single-cell transcriptome atlas of the mouse epididymis and revealed the novel distribution pattern of mitochondria and key genes that may be linked to sperm functionalities in the first wave and subsequent wave of sperm, providing a roadmap to be emulated in efforts to achieve sperm maturation regulation in the epididymis.