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Alternative splicing (AS) is a ubiquitous phenomenon among eukaryotic intron-containing genes, which greatly contributes to transcriptome and proteome diversity. Here we performed the isoform sequencing (Iso-Seq) of soybean underground tissues inoculated and uninoculated with Rhizobium and obtained 200,681 full-length transcripts covering 26,183 gene loci. It was found that 80.78% of the multi-exon loci produced more than one splicing variant. Comprehensive analysis of these identified 7874 differentially splicing events with highly diverse splicing patterns during nodule development, especially in defense and transport-related processes. We further profiled genes with differential isoform usage and revealed that 2008 multi-isoform loci underwent stage-specific or simultaneous major isoform switches after Rhizobium inoculation, indicating that AS is a vital way to regulate nodule development. Moreover, we took the lead in identifying 1563 high-confidence long non-coding RNAs (lncRNAs) in soybean, and 157 of them are differentially expressed during nodule development. Therefore, our study uncovers the landscape of AS during the soybean-<i>Rhizobium</i> interaction and provides systematic transcriptomic data for future study of multiple novel directions in soybean.