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Background: Natural antisense transcripts (NATs) are a class of RNAs that contain a sequence complementary to other transcripts. NATs occur widely in eukaryotes and play critical roles in post-transcriptional regulation. Soybean NAT sequences are predicted in the PlantNATsDB, but detailed analyses of these NATs remain to be performed. Results: A total of 26,216 NATs, including 994 cis-NATs and 25,222 trans-NATs, were predicted in soybean. Each sense transcript had 1-177 antisense transcripts. We identified 21 trans-NATs using RT-PCR amplification. Additionally, we identified 179 cis-NATs and 6,629 trans-NATs that gave rise to small RNAs; these were enriched in the NAT overlapping region. The most abundant small RNAs were 21, 22, and 24 nt in length. The generation of small RNAs was biased to one stand of the NATs, and the degradation of NATs was biased. High-throughput sequencing of the degradome allowed for the global identification of NAT small interfering RNAs (nat-siRNAs) targets. 446 target genes for 165 of these nat-siRNAs were identified. The nat-siRNA target could be one transcript of a given NAT, or from other gene transcripts. We identified five NAT transcripts containing a hairpin structure that is characteristic of pre-miRNA. We identified a total of 86 microRNA (miRNA) targets that had antisense transcripts in soybean. Conclusions: We globally identified nat-siRNAs, and the targets of nat-siRNAs in soybean. It is likely that the cis-NATs, trans-NATs, nat-siRNAs, miRNAs, and miRNA targets form complex regulatory networks. [ABSTRACT FROM AUTHOR]