We identified two splicing variants of GhLSM1B (GhLSM1BS and GhLSM1BL) with distinct expression patterns and predicted 3D structures, despite sharing the same nuclear localization. Overexpression of GhLSM1BS, but not GhLSM1BL, accelerated cotton callus proliferation and altered cell morphology during somatic embryogenesis, accompanied by altered expression of CYP450 family genes and elevated brassinosteroid levels.