Abstract: The intestinal microflora influences the overall health and immune status of poultry birds. It is therefore obvious that the future success of poultry farming lies in the use of probiotics as growth promoters and health management agents. A total of 196 points of lay (12 weeks) hens ISA strain of pullets was used to investigate the effects of Pichia kudriavzevii on the immunological response and ceacal microflora of layer hens. The birds were randomly allotted to seven treatment groups of 9 replicates each. There were 3 birds per replicate, making a total of 27 birds per treatment in a completely randomized design. The birds were vaccinated with Lasota strain of New Castle Disease Vaccine (NCDV) at week 20 of diet supplementation. At the end of the experiment, the spleen of birds was collected for gene expression of some cytokines. The result of this study showed (P<0.05) and a sustained antibody titre against NCDV in all the treatment groups supplemented with strains of P. kudriavzevii as compared with the positive and negative control groups. Interferon-gamma and interleukin 10 gene expressions in the spleen was upregulated by the yeast strains with the highest IFN-γ concentration of 34.38 µg/mL recorded in diet supplemented with P. kudriavzevii MH458239. It was also observed that total bacteria count was significantly (P<0.05) reduced through the administration of these yeast strains. Conclusively, the use of P. kudriavzevii as a growth promoter in layer hen positively influenced their caecal microflora which consequently primed their immune response.