Simple SummaryCold is an important limiting factor in the development of the livestock and poultry industry, which has adverse effects on the intestinal barrier and immune function of animals. While appropriate cold stimulation can improve immune function, it remains unclear whether intermittent and mild cold stimulation can improve production performance, intestinal barrier function and stress tolerance through improving immunoglobulins and intestinal barrier gene levels. In this study, broilers were subjected to intermittent and mild cold stimulation and acute cold stress to assess its effects on production performance, intestinal barrier and immune function. The results indicate that intermittent and mild cold stimulation can improve the production performance, intestinal barrier and acute cold stress resistance of broilers through modulating the levels of intestinal barrier genes and immunoglobulins. We hope to provide a theoretical basis for the development of husbandry in cold environments.AbstractIn order to improve the adaptability of broilers to low-temperature environments and their ability to resist acute cold stress (ACS), 240 one-day-old broilers were selected and randomly divided into three groups. The control treatment (CC) group was raised at the conventional feeding temperature from 1–43 days (d), the cold stimulation treatment (CS) group was kept at 3 °C below the temperature of CC at 1 d intervals for 3 and 6 h from 15 to 35 d, namely, CS3 and CS6, respectively. Then, all broilers were kept at 20 °C from 36 to 43 d. ACS was then carried out at 44 d, and the ambient temperature was dropped to 10 °C for 6 h. The study investigated the production performance, as well as levels of intestinal barrier genes (includingClaudin-1,E-cadherin,Occludin,ZO-1,ZO-2andMucin2), secretory IgA in duodenum and jejunum, and immunoglobulins (IgA and IgG) in serum. The results showed that IMCS could increase the daily weight gain and decrease the feed conversion ratio. During IMCS, the expression levels of intestinal barrier genes were up-regulated and the content of secretory IgA was increased. When IMCS ceased for one week, the level of immunoglobulins in serum stabilized, and the expression levels ofOccludin,ZO-2andMucin2still maintained high levels. After ACS, broilers that received IMCS training maintained high levels of intestinal barrier genes and secretory IgA.