Arsenic (As) is widely distributed in our living environment and is useful for industry, agriculture, medical treatment, and other fields. Arsenic trioxide (As2O3) is an existing form of As. Exposure to As2O3 has a toxic effect on humans and animals. It not only leads to skin cancer, peripheral vascular disease, hyperkeratosis, etc. but also interferes with the functioning of the gastrointestinal tract. The gastrointestinal tract is an important organ for animals to transform the food they eat into the nutrients their body needs for maintenance and growth. Heat shock proteins (Hsps) exist in the non-stress normal cells and their expression increases under stimuli. Therefore, we wonder whether the "stimulus" of As2O3 could change the messenger RNA (mRNA) abundance and expression level of Hsps in the gastrointestinal tract of birds. To investigate the relation between arseniasis and Hsp alterations in the chicken's gastrointestinal tract induced by an As2O3-supplemented diet, we selected 72 one-day-old male Hy-line chickens and randomly divided them into four groups. They were fed either a commercial diet or an As2O3-supplemented diet containing 7.5, 15, and 30 mg/kg As2O3. The experiment lasted for 90 days, and gastrointestinal tract tissue samples (gizzard, glandular stomach, duodenum, jejunum, ileum, cecum, and rectum) were collected at 30, 60, and 90 days. The mRNA contents of Hsps (including Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90) were examined by real-time PCR (RT-PCR). The correlation between As2O3 and Hsp genes was assessed. In addition, the protein expression levels of Hsp60 and Hsp70 in the gastrointestinal tract tissue samples were measured by western blot. The results indicated that the mRNA expression levels and the Hsp expression levels in the gastrointestinal tract tissues of chickens with As2O3 supplementation increased at different time points in a dose-dependent manner (p < 0.05 or p < 0.01). These data suggested that arseniasis influenced the mRNA abundance of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 and the protein expression levels of Hsp60 and Hsp70 in the chicken's gastrointestinal tract tissues.