1-aminocyclopropane-1-carboxylate (ACC) synthase is the rate-limiting enzyme in ethylene biosynthesis pathway in plants, which catalyzes the conversion from S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylate (ACC). The ACC synthase (ACS) is encoded by a multiple gene family and its expression is differentially regulated by a complicated network composed of biotic and abiotic signals responding to both internal and external stimuli under the translational and posttranslational levels. However, its expression regulatory mechanism now still remains incompletely clear. Recent years, several researches showed that the Natural Antisense Transcripts (NATs) played a critical role in gene expression regulation in eukaryotic organism, which attracted increasing attentions. Recently, a putative ACC synthase cis–NAT was predicted in Arabidopisis by the genomic-wide scanning technique. In this study, a cis-natural antisense transcript (cis-NATs) of ACC synthase gene, named ASACS2, was identified from six northeastern soybean cultivars of China by RT-PCR and bidirectional sequencing. Further investigation by Real-time RT-PCR showed that during the vegetative growth stage ASACS2 co-existed with its higher-abundant sense counterparts, SACS2. Interestingly, Real-time RT-PCR also showed that the ratio between ASACS2 and SACS2 remain constant in each cultivar, but varied among six cultivars, suggesting the cultivar specificity. This study indicates that ASACS2 might be a potential regulatory actor in ethylene metabolism pathway. The further knowledge of this actor would facilitate us to better understand the gene regulation machinery in plants and the application in transgenic research.