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Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and biotic and abiotic stresses. Recent work showed that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various AS small-molecule inhibitors that perturb splicing and thereby provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Here, I show that the macrolide Pladienolide B (PB) and herboxidiene (GEX1A) inhibits both constitutive and alternative splicing, mimics an abiotic stress signal, and activates the abscisic acid (ABA) pathway in plants. Moreover, PB and GEX1A activate genome-wide transcriptional patterns involved in abiotic stress responses in plants. PB and GEX1A treatment triggered the ABA signaling pathway, activated ABA-inducible promoters, and led to stomatal closure. Interestingly, PB and GEX1A elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. This work establishes PB and GEX1A as potent splicing inhibitors in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.