Larvae of the goldenrod gall fly, Eurosta solidaginis, can survive winter temperatures as cold as –55oC by using the freeze tolerance strategy of cold hardiness. This study examines pyruvate kinase (PK) and glycerol-3-phosphate dehydrogenase (G3PDH) that were purified from gall fly larvae acclimated to 5oC (control) and −15oC (freeze-exposed). The roles that these enzymes play in polyol cryoprotectant synthesis and metabolic regulation were investigated. A primary focus was the potential for reversible post-translational modifications (PTMs) to regulate enzyme activity. Several PTMs were analyzed for PK and G3PDH from control and freeze-exposed gall fly larvae using dot-blot analysis. Results showed that low and high phosphate forms of each enzyme exist and these differ in kinetic properties, G3PDH also showed different levels of ubiquitination. Changes in substrate affinities and different interactions with endogenous sugars suggest that PK activity is maintained whereas G3PDH is inhibited during freezing.