Ischemic stroke (IS) prevalence rising annually, the necessity of discovering non-interventional genetic influences is progressing. Single nucleotide polymorphism (SNP) plays a pivotal role in stable inheritance of disease susceptibility. Based on the relationship between Alpha- Kinase 1 (ALPK1) and traditional IS risk factors especially hyperuricemia, our study investigated the association and function of ALPK1 SNPs with IS susceptibility.A case-control study of 1539 patients and 933 controls from northeast China was conducted. Genotyping information of ALPK1 rs2074379 and rs2074388 was collected. Four types of plasmids including rs2074379/rs2074388 G/G, A/G, G/A, and A/A were transfected into 293T cells to observe ALPK1 and SLC22A12 expression. Possible ALPK1 structures of different SNPs were predicted online.Genotype GG (OR = 1.371, CI = 1.029-1.828, P = 0.031) and GA (OR = 1.326, CI = 1.110-1.584, P = 0.002) of rs2074379 and GA of rs2074388 (OR = 1.359, CI = 1.137-1.624, P = 0.001) were found significantly susceptible to IS, with G allele on sites to be a risk allele. Rs2074379 had a multiplicative interaction with hyperuricemia (OR = 1.637, CI = 1.157-2.315, P = 0.005). Uric acid levels differed in genotypes (P < 0.001). The expression of ALPK1 (P < 0.01) and SLC22A12 in membrane urate transporter 1 (URAT1) protein (P < 0.05) functionally changed with G allele on either site. With glycine changing into aspartic acid at rs2074388, the protein secondary structure changed, but the ALPK1 protein subtype remained still.ALPK1 rs2074379 and rs2074388 SNPs were functionally associated with IS susceptibility. The wild allele progressed IS risk probably by reducing ALPK1 expression and inhibiting URAT1 raising the uric acid level, contributing to further exploration of pathogenetic mechanisms of stroke. Chinese Clinical Trial Registration number: ChiCTR-COC-17013559.Copyright © 2024. Published by Elsevier B.V.