High temperatures jeopardize animal welfare of dairy cows. During periods of hot weather, leaky gut syndrome, characterized by impaired gut integrity, may occur. Diminished intestinal barrier facilitates penetration of toxic and bacterial compounds, leading to increased inflammation in jejunum and reflected by increased appearance of immune cells. This study aimed at identification of immune cell populations infiltrated in the lamina propria and underneath the muscularis mucosae of jejunum of heat-stressed dairy cows. Three German Holstein dairy cows (240 ± 25 d in milk) were exposed to 28°C (52 ± 2% RH; THI = 76) for 4 d, with ad libitum feeding tempered to 28°C, to induce heat stress (HS). Cows were then slaughtered to obtain jejunum samples. Infiltrated cells were collected from dehydrated cryo-sections by laser microdissection and RNA was extracted. RNaseq libraries were prepared with NuGEN’s Ovation Solo RNA-Seq Kit and sequenced on the Illumina HiSeq2500 platform using 2x100bp paired-end sequencing cycles. The resulting reads were checked for quality, mapped to the bovine reference genome with HISAT2 and analyzed using IG viewer. Mucosa from a cow kept under thermoneutrality and ad libitum feeding served as control. Immunohistochemistry was used to detect CD3, CR2, CD163 and SIRPA in serial sections of jejunum. The dendritic cell marker SIRPA was detected in all samples. Macrophage-related genes including ITGAM, CD14, FCGR3A, CD68, and MRC1 were also expressed in all samples, whereas CD163 was only present in cells from HS cows but not in control. Furthermore, the B cell marker CR2, and T cell markers CD4 and CD3E were found in all samples. Immunofluorescence confirmed that infiltrated cells were CD163⁺ and SIRPA⁺; but CD3⁺ and CR2⁺ cells were only detected in different regions. Heat-stress induced infiltration of immune cells in the lamina propria and underneath the muscularis mucosae. Immune cells were identified as macrophages and dendritic cells in concordance between RNaseq and immunohistochemistry results, whereas expression of markers for B and T cells could not be confirmed on protein level.