Background Gestational hypothyroidism (G-HypoT) is one of the most common thyroid diseases in pregnant women. Human milk, which closely links the mother with infant, is an important factor to the infant health. Here, we analyzed the colostrum whey proteome of women with or without G-HypoT. Methods and results Using high-mass accuracy and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), 1055 proteins were identified. Tandem Mass Tags (TMT) analysis identified differentially expressed proteins between G-HypoT and non-G-HypoT mothers. Of 44 proteins identified, 15 proteins were significantly increased in G-HypoT colostrum whey, while 29 were significantly decreased. Analysis revealed that enzymes involved in carbohydrate metabolism, and that reflect the metabolic activities in breastfeeding women, including fructose-1, 6-bisphosphatase 1, phosphoglycerate mutase 1 were down-regulated. Cell structural proteins, biomarkers of mammary integrity development, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were lower in G-HypoT colostrum whey. However, immune protein fragments like Ig gamma-3 chain C region increased in G-HypoT colostrum whey. Conclusion These results implied that G-HypoT may changed human colostrum whey protein in composition level, decreasing levels of metabolic proteins and cell-structure proteins, while increasing levels of immune-related proteins, which may compromise or reflect mothers' and infants' health.