Recent research has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis can modulate the subsequent phases of the process, as well as the behaviour of the resulting plants ex vitro. In this sense, the development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental temperature changes. As a result, in this study, the proteome of radiata pine somatic embryos was studies by LC-MS after the application of high temperatures during induction of embryonal masses (23 °C, 8 weeks, control, 40 °C, 4 h, 60 °C, 5 min). Then, the content of specific soluble sugars and sugar alcohols by HPLC was analysed. Results confirmed a decreased initiation rate of embryonal masses under high temperatures and a slight increase in the production of somatic embryos, as observed in previous studies. Besides, heat provoked a long-term readjustment of the protein synthesis machinery, by regulating translation and the composition of ribosomes. The levels of proteins related to methylation and posttranscriptional regulation mechanisms were also altered under high temperatures, coupled with higher contents of heat shock proteins and chaperones, transmembrane transport proteins, enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol), and changes in the composition of cell-wall carbohydrates. These modifications occurred at the expense of the synthesis of molecules involved in oxidative stress responses, such as peroxidases, thiamine, phenolic compounds and some amino acids, by a decreased activity of the glycolytic pathway and reduced nitrogen assimilation.