The gene cluster for spectinomycin biosynthesis from Streptomyces spectabilis was analyzed completely and registered under the accession number EU255259 at the National Center for Biotechnology Information. Based on sequence analysis, spcM of the S. spectabilis cluster is the only methyltransferase candidate required for methylation in spectinomycin biosynthesis. It has high similarity with the conserved domain of DNA methylase, which contains both N-4 cytosine-specific DNA methylases and N-6 adenine-specific DNA methylases. Nucleotide methylation can provide antibiotic resistance, such as 16S rRNA methyltransferase, to Enterobacteriaceae. We therefore tested a hypothesis that SpcM offers aminoglycoside resistance to bacteria. The heterologous expression of spcM in Escherichia coli and S. lividans enhanced resistance against spectinomycin and its relative aminoglycoside antibiotics. We therefore propose that one of the functions of SpcM may be conferring aminoglycoside antibiotic resistance to cells.