Glucose-6-phosphate dehydrogenase deficiency (G6PD) also known as favism anemia is an enzyme deficiency condition caused by mutations in the G6PD gene that is a prevalent cause of hemolytic anemia in humans. This defect is an X-linked recessive illness, males are frequently affected while females are carriers of the defect. G6PD enzyme protects the red blood cell (RBC) against harmful substances that cause hemolysis. To assess mothers of children and pregnant women’s knowledge, attitude, and practice toward glucose-6-phosphate dehydrogenase deficiency in Saudi Arabia. The study population was consisting of Saudi mothers n= 889 aged from 18 years to 55 years old. The cross-sectional online questionnaire was carried out from September 2022 to November 2023 in the Kingdom of Saudi Arabia. The study included 859 participants, 91.5% were females and 8.5% were males. 10.4% of participants have a family history of G6PD. 54.7% heard of G6PD. 18.2% of participants reported that the presence of G6PD in the family was a prerequisite for its occurrence. 39% reported that pallor is a sign of G6PD. 65% of participants had good knowledge scores and 35% had poor knowledge scores. Attitude score shows that 55% of participants had negative attitudes and 45% had positive attitudes. Practice score was reported as 15.8% had a good practice and 84.2% had bad practice. The study shows that the Saudi population had generally poor knowledge, attitude, and practice toward G6PD. Knowledge score of G6PD was significantly associated with gender, educational level, and residency region in Saudi Arabia.