Splenectomy is a surgery indicated in case of splenic rupture after injury, when there are tumors in the spleen, or as a treatment for certain diseases, such as idiopathic thrombocytopenic purpura and spherocytosis. The aims of the study were to assess the immunological response to the Haemophilus influenzae type b (Hib) vaccine and the post-vaccination changes in lymphocyte subsets and cell activation markers in splenectomized patients and healthy volunteers. Blood samples were collected from 25 patients that had undergone splenectomy and from 15 healthy, non-splenectomized volunteers. All participants received a single dose of Hib vaccine. The concentration of specific Hib antibodies was assessed by an enzyme-linked immunosorbent assay. Selected immune cell populations were evaluated using flow cytometry. The analysis of the antibody titers against Hib showed statistically significant differences in both groups. There was a significantly higher percentage (p = 0.0012) and absolute value (p = 0.0003) of natural killer T (NKT)-like cells (CD3+/CD16+ CD56+) in the study group, compared to the control group. The levels of natural killer (NK) and NKT cells did not change relative to the cause and age of splenectomy. The quantity and percentage of regulatory T (Treg) cells were higher in the study group compared to the control group (p < 0.0001). No significant correlations were found between the time elapsed since splenectomy, the age of the patients, and the Treg levels. Our study showed that spleen resection results in an important deterioration of Treg cells and Th17 cell balance which may contribute to an incomplete immunological response.