BACKGROUND: Echinacanthus longipes is an endemic species in the Sino-Vietnamese karst flora in the family Acanthaceae. It displays distinctive environmental adaptation characteristics in karst regions. Although it provides an important model for understanding the role of limestone karst in speciation and endemism, the mitochondrial genome (mtDNA) of E. longipes has not been fully characterized. RESULTS: Here, the mtDNA of E. longipes was successfully assembled as a complex structure in the form of two small circular and three linear molecules with a total length of 810,200 bp. The annotated results revealed 36 protein-coding genes (PCGs), 22 tRNA genes, and three rRNA genes in this mtDNA. Notably, substantial sequence repeats and more tRNAs translocations from the chloroplast to the mtDNA were identified. Among the PCGs of E. longipes, the majority of 401 RNA editing sites were involved in amino acid transitions to hydrophobic sites. The current phylogenetic analysis based on PCGs revealed the evolution of Lamiales and a close relationship between E. longipes and Avicennia marina. However, comparative analyses, including size, structure, GC contents, and genes, reflected the variation in the mitogenomes within Acanthaceae, and the collinearity analysis confirmed the low level of conservation in the genomes of related species in Lamiales. Moreover, the Ka/Ks analysis revealed that negative selection occurred on most PCGs, with the notable exception of ccmB, which underwent positive selection. Interestingly, the ccmB gene had the most protein editing sites. CONCLUSIONS: This study will be invaluable for the mitochondrial study of Acanthaceae. It also provides extensive information for functional genetic and adaptive studies of Echinacanthus in karst regions in the future.