N⁶-methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m⁶A as well as m⁶A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m⁶A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m⁶A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m⁶A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m⁶A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m⁶A pattern that is distinct from those in eukaryotes. Most m⁶A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m⁶A peaks indicates that the majority of m⁶A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m⁶A in these pathways.