A total number of 720 turkey hatching eggs with an average weight of 81± 1g were used to study the effect of injecting the eggs with pyridoxine (vitamin B₆) at 10^th, 17^th and 24^th days of incubation. Hatching eggs were divided into equal six groups at each injecting time. The 1^st group without injection or pierced was served as control 1, the 2^nd group without injection was pierced in the blunt end and served as control 2, while eggs of the 3^rd group was injected into amniotic sac through the air sac with 0.2 ml saline solution. The eggs of the 4^th, 5^th and 6^th groups were injected as group 3 with 400, 600 and 800 μg pyridoxine, respectively, dissolved in 0.2 ml saline solution.  The results obtained could be summarized as follows: Embryonic mortality percentage (EM) as well as hatchability percentage (H) and chick weight at hatch (CWH) showed significant (P≤0.05) differences among injection times of embryonic development (ED). The best EM, H and CWH values were recorded at the10^th ED. Injection of 0.2 ml saline solution with different levels of pyridoxine to the hatching eggs, significantly (P≤0.05) decrease the EM and significantly (P≤0.05) improved both H and CWH. The best value of Albumin (A)/globulin (G) ratio was at the 17^th and 24^th ED compared to 10^th ED. The best values of plasma total protein (PTP), globulin (G) and A/G ratio were recorded by eggs injected with the lowest level of pyridoxine (400µg). Moreover, the best value of A/G ratio was recorded by control group at the 24^th ED. The best value of plasma cholesterol level was recorded for eggs injected the highest level of pyridoxine (800µg) at the 10^th ED. Plasma concentrations of thyroid hormones were significantly increased (P≤0.05) by increasing pyridoxine injection. On the other hand, insignificant differences were noted in plasma thyroid hormones concentrations due to periods of ED. The highest values of T₃ hormone were recorded by eggs injected the highest level of pyridoxine at both the 24^th and 17^th ED, respectively. While the highest values of T₄ hormone were recorded by eggs injected the highest level of pyridoxine at both the 17^th and 10^th ED, respectively. In conclusion, in-ovo pyridoxine injection with 800µg to turkey eggs at 10 and 17 day of incubation is a practical and beneficial procedure to improve the hatchability traits