<p>ssSomatic cell fusion breeding involves simultaneously transferring cytoplasmic and nuclear genes to create new germplasm resources. However, this technique is difficult to apply in some heterokaryon-derived cells, such as sugarcane cells, due to the limited understanding of physiological and molecular mechanisms in the heterokaryon-derived cells post-fusion. Flow cytometry (FCM) was used to screen heterokaryon-derived cells, orthogonal design was used to screen the conditions for culture, and cytology, proteomics and gene expression analyses were performed to test the regenerative capacity of the heterokaryon-derived cells of sugarcane. The orthogonal experiment results show that the best and worst medium hormone combinations for regeneration rate of heterokaryon-derived cells were A₂B₃ (2,4-D 2 mg/L+6-BA 3 mg/L) and A₃B₁ (2,4-D 4 mg/L+6-BA 1 mg/L), respectively. The best hormone combination A₂B₃ combination upregulated key regeneration genes (GAUT, CESA, CyclinA/B/D3, Cdc2, PSK), promoted microtubule rearrangement, and accelerated cell-wall regeneration and division. The most abundant differential proteins before and after culture in the A₂B₃ were mainly involved in RNA processing and modification, cell-cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Our findings contribute to the understanding of the molecular mechanism underlying hormone-regulated regeneration of sugarcane heterokaryon-derived cells.</p> Graphical Abstract <p></p>