Background: The ability of hemoglobin to bind and dissociate oxygen is crucial in delivering oxygen to tissues and is influenced by a range of physiological states, compensatory mechanisms, and pathological conditions. This may be illustrated by the oxyhemoglobin dissociation curve (ODC). The key parameter for evaluating the oxygen affinity to hemoglobin is p50. The aim of this study was to evaluate the impact of hemodialysis on p50 in a group of patients with chronic kidney disease (CKD). An additional goal was to assess the correlation between p50 and the parameters of erythropoiesis, point-of-care testing (POCT), and other laboratory parameters. Methods: One hundred and eighty patients (106 male, 74 female), mean age 62.5 ± 17 years, with CKD stage G4 and G5 were enrolled in this cross-sectional study. Patients were divided into two groups, including 65 hemodialysis (HD) patients and 115 patients not receiving dialysis (non-HD). During the standard procedure of arteriovenous fistula creation, blood samples from the artery (A) and the vein (V) were taken for POCT. The causes of CKD, as well as demographic and comorbidity data, were obtained from medical records and direct interviews. Results: The weekly dose of erythropoietin was higher in HD patients than in non-HD patients (4914 ± 2253 UI vs. 403 ± 798 UI, p < 0.01), but hemoglobin levels did not differ between these groups. In the group of non-HD patients, more advanced metabolic acidosis (MA) was found, compared to the group with HD. In arterial and venosus blood samples, the non-HD group had significantly lower pH, pCO2 and HCO3−. This group had a higher proportion of individuals with MA with HCO3− < 22 mmol/L (42% vs. 24%, p < 0.01). The absolute difference of p50 in arterial and venous blood was determined using the formula Δp50 = (p50-A) − (p50-V). Δp50 was significantly higher in the HD group in comparison to non-HD (0.08 ± 2.05 mmHg vs. −0.66 ± 1.93 mmHg, p = 0,02). There was a negative correlation between pH and the p50 value in arterial (pH-A vs. p50-A, r = −0.56, p < 0.01) and venous blood (pH-V vs. p50-V, r = −0.45, p < 0.01). In non-HD patients, hemoglobin levels correlated negatively with p50 (r = −0.29, p < 0.01), whereas no significant relation was found in HD patients. Conclusions: The ODC in pre-dialysis CKD (non-HD) patients is shifted to the right due to MA, and this is an additional factor influencing erythropoiesis. Hemodialysis restores the natural differences in hemoglobin’s dissociation characteristics in the arterial and venous circulation.