Human leukocyte antigen-G (HLA-G) is linked to the development of human malignancies via immune escape mechanisms. The chief variations for HLA-G were found in three prime untranslated regions (3'UTR). The current study aims to evaluate the distribution of HLA-G rs1063320 (G+3142 C>G), HLA-G*01:03, and HLA-G*01:05N polymorphisms with risk of acute lymphocytic leukemia (ALL) in Saudi Arabia. This case-control study analyzed 232 samples from 117 patients with ALL and 115 healthy controls (HCN) using the PCR-RFLP method. Associations between HLA-G and ALL risk were analyzed using allele contrasts. The HLAG rs1063320 G+3142 C>G polymorphism results showed a reduced risk of ALL in the dominant G/C model odds ratios (OR) = 0.34, 95% confidence interval (CI) = 0.12-0.98, P = 0.041), stratified by age. However, those stratified by gender, showed decreased risk of ALL in all genetic inheritance models tested: codominant model C/G versus G/G (OR = 0.24, 95% CI = 0.06-0.99), C/C versus G/G (OR = 0.01, 95% CI = 0.00-0.12), P = 0.0001), and dominant model C/G-C/C versus G/G (OR = 0.12, 95% CI = 0.03-0.47, P = 0.000004), and the recessive model C/C versus G/G-C/G (OR = 0.03, 95% CI = 0.00-0.24, P = 0.0001), log-additive (OR = 0.12, 95% CI = 0.04-0.35, P = 0.0001). Conversely, the G*01:03 allele was not found in ALL or HCN, whereas the G*01:05N allele showed polymorphic frequencies that were not significant. In conclusion, the HLA-G +3142 C>G polymorphism significantly decreased the prevalence of ALL stratified by gender and age polymorphisms in the risk of ALL in the pediatric Saudi population.