Granulosa cell apoptosis contributes to the occurrence of diminished ovarian reserve (DOR). HOXA1, belonging to the HOX gene family, is involved in regulating cancer cell apoptosis. However, whether HOXA1 participates in the granulosa cell apoptosis in DOR patients remains to be elucidated. In the current study, we demonstrated the differential transcriptomic landscape of granulosa cells in DOR patients compared to that in the controls and identified decreased expression of the HOXA1 gene. Meanwhile, we found that HOXA1 was a gonadotropin-response gene, in which FSH could promote its expression, whereas LH inhibited HOXA1 expression in human granulosa cells. CCK-8 assay, flow cytometry and TUNEL staining results showed that inhibition of endogenous HOXA1 expression promoted human granulosa cell apoptosis. Moreover, knockdown of HOXA1 increased Bax while reducing Bcl2 protein expression. Furthermore, we found a total of 947 differentially expressed genes (DEGs), including 426 upregulated genes and 521 downregulated genes using transcriptome sequencing technology. Enrichment analysis results showed that the DEGs were involved in apoptosis and mitochondrial function-related signaling pathways. Knockdown of HOXA1 impaired mitochondrial functions, exhibiting increased reactive oxygen species (ROS) and cytoplasmic Ca levels, decreased mitochondrial membrane potential, ATP production and mitochondrial DNA (mtDNA) copy number, and abnormal mitochondrial cristae. Our findings demonstrated that aberrantly reduced HOXA1 expression induced granulosa cell apoptosis in DOR patients and impaired mitochondrial function, which highlighted the potential role of HOXA1 in the occurrence of DOR and provided new insight for the treatment of DOR.