In somatic embryogenesis, embryos are formed from a single or small group of somatic cells in response to stress and hormonal stimuli. In this study, a high-throughput phosphoproteomic analysis was used to identify and quantify phosphoproteins related to the acquisition of embryogenic competence in sugarcane. Embryogenic (EC) and nonembryogenic (NEC) calli were compared at the multiplication phase, resulting in the identification of 1279 phosphoproteins. After the differential accumulation analysis, 163 phosphoproteins were identified as unique to EC, and nine were unique to NEC, while 51 were upaccumulated, and 40 were downaccumulated in EC compared to NEC. These phosphopeptides were mainly single phosphorylated peptides, with serine residues representing the major site of phosphorylation. The motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx] and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. Gene ontology enrichment revealed an overrepresentation of biological processes involved in embryonic development, showing that EC-related phosphoproteins (unique to EC and upaccumulated) were associated with stress responses, regulation of gene expression and epigenetic modifications. The NEC-related phosphoproteins (unique to NEC and downaccumulated) were associated with the translation process. In this sense, EC-related phosphoproteins described as potential regulators of abiotic stress tolerance in response to ABA-induced signals such as OSK3 (a catalytic subunit of SnRK1), ABF1, LEAs, REM4.1 and RD29Bs were identified. On the other hand, the NEC-related phosphoproteins EDR1 and PP2Ac-2 were negative regulators of ABA, suggesting a role of ABA in the acquisition of embryogenic competence linked to stress tolerance. Moreover, EC-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TPL, as well as those involved in embryo development, including ASIL1, M3KE1, MCM2, and GRV2, were identified as putative potential regulators of embryogenic competence. Our results provide novel data that may help elucidate the phosphorylation mechanisms controlling the activity, affinity and localization of specific proteins during somatic embryogenesis and suggest a role for these proteins in the hormonal signalling cascade leading to stress adaptation by reprogramming genetic expression.