Abstract: The Hedgehog signaling pathway participates in the occurrence and progression of cancers including gastric cancer. We conducted this study to evaluate whether genetic variants in the Hedgehog signaling pathway genes would affect gastric cancer risk. Multi-marker Analysis of GenoMic Annotation (MAGMA) was used to investigate the aggregated genetic effects of single nucleotide polymorphisms (SNPs) assigned to candidate genes. The relationship between SNPs and gastric cancer risk was estimated by multivariate logistic regression analyses. Gene expression was calculated using databases obtained from The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO). Kaplan‐Meier plotter was used to evaluate the association between gene expression with gastric cancer survival. Tumor Immune Estimation Resource 2.0 (TIMER 2.0) was applied to determine the correlation between selected gene expression and the immune cell infiltration degree. We identified that the G allele of rs2990912 in KIF27 was associated with higher gastric cancer risk, especially in the young and male subgroups. The expression of KIF27 in gastric cancer tissues was higher than that in normal tissues, leading to poor survival in gastric cancer patients. Besides, KIF27 expression was related to immune cell infiltration and positively correlated with PD-L1 expression. Our findings highlight the key role of genetic variation in the Hedgehog signaling pathway genes in gastric cancer susceptibility, which may provide important insights into the diagnosis, prognosis, and treatment of gastric cancer.