This study determined the effects of pro-inflammatory cytokines (interleukin (IL)-1β and IL-6) on the expression of eggshell mineralization-related ion transporters in the hen uterus mucosa. Uterine mucosal tissues collected from White Leghorn laying hens were cultured for 1.5 or 3 h in TCM-199 medium with or without 100 ng/mL recombinant chicken IL-1β or IL-6. Total RNA and protein were extracted from the cultured tissues for real-time polymerase chain reaction (PCR) and western blot analyses and some tissues were processed into paraffin sections for immunostaining with calcium-binding protein D28K (CaBP-D28K) antibody. The gene expression of CaBP-D28K, PMCA1, PMCA2 (plasma membrane calcium-transporting ATPase 1 and 2; calcium pumps), CA2 (carbonic anhydrase 2), and SLC26A9 (solute carrier family 26 member 9; HCO₃⁻ transporter) was analyzed by real-time PCR and protein density of CaBP-D28K by western blotting. Expression of CaBP-D28K, PMCA1, PMCA2, CA2, and SLC26A9 was significantly higher in the tissues treated with IL-1β and IL-6 than in the control group at 1.5 h of incubation. Immunoreactive CaBP-D28K was localized in the uterine tubular gland cells in all groups, but its level was significantly lower in the tissues incubated for 1.5 h with IL-1β and IL-6 than in the control group. No significant differences were observed in the expression of all tested genes and CaBP-D28k content between the cytokine-treated and control groups at 3 h of incubation. These results suggest that IL-1β and IL-6 may not suppress the expression of genes related to Ca²⁺ and HCO3⁻ transportation for eggshell formation, while CaBP-D28K protein content in uterine glandular cells was reduced by these cytokines during the early exposure phase. Thus, IL-1β and IL-6 induced by infections may disrupt the transportation of Ca²⁺ for eggshell formation through decreased CaBP-D28K content in the uterus.