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In the present study, electrophoretic mobility shift assays were used to identify temperature responsive elements in the 5′ upstream region (5′ UTR) of the Spirulina desD gene. Overlapping, synthetic oligonucleotides of both sense and anti-sense strands that spanned the entire 5′ UTR of the gene were analyzed. The responsive DNA-binding protein complexes were identified using liquid chromatography–tandem mass spectrometry. The results indicated that the cold-responsive elements were located at –453 to –247, –197 to –151, –105 to –76, and –50 to –1, whereas the low-temperature specific regulatory regions were located at –372 to –352. Moreover, the heat-responsive elements were located at –347 to –243, –197 to –151, and –124 to –1, whereas the high-temperature specific elements were located between –130 to –101 and –30 to –1. In terms of regulatory protein complexes under the two stress conditions, Trx was only detected in the low-temperature responsive protein complex, and divalent cations were essential for the binding of the protein complex to the regulatory elements. Furthermore, Trx was shown to play a critical role as a reducing agent that inactivates the Spirulina desD repressor, GntR. Consequently, the desD gene expression is induced under the low-temperature condition.