This study offers proteomic elucidation of heat pretreatment-induced alleviation of UV-B toxicity in Anabaena doliolum. Heat-pretreated cells exposed to UV-B showed improved activity of PSI, PSII, whole chain, 14C fixation, ATP and NADPH contents compared to UV-B alone. Proteomic analysis using two-dimensional gel electrophoresis (2-DE), MALDI-TOF MS/MS and reverse transcription polymerase chain reaction (RT-PCR) of UV-B and heat pretreatment followed by UV-B–treated cells exhibited significant and reproducible alterations in nine proteins homologous to phycocyanin-α-chain (PC-α-chain), phycoerythrocyanin-α-chain (PEC-α-chain), hypothetical protein alr0882, phycobilisome core component (PBS-CC), iron superoxide dismutase (Fe-SOD), fructose-1,6-bisphosphate aldolase (FBA), nucleoside diphosphate kinase (NDPK), phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) large chain. Except the PEC-α-chain, hypothetical protein alr0882 and PBS-CC, all other proteins showed upregulation at low doses of UV-B (U2) and significant downregulation at higher doses of UV-B (U5). The disruption of redox status, signaling, pentose phosphate pathway and Calvin cycle appears to be due to the downregulation of Fe-SOD, NDPK, FBA, PRK and RuBisCo thereby leading to the death of Anabaena. In contrast to this, the upregulation of all the above proteins in heat-pretreated cells, harboring different heat shock proteins (HSPs) like 60, 26 and 16.6, followed by UV-B treatment than only the UV-B–treated ones suggests a protective role of HSPs in mitigating UV-B toxicity.