Rumination time (RT) in dairy cattle is a crucial indicator of health, production, reproduction, and greenhouse gas emissions. With moderate heritability estimates for RT, there is potential for further analyses regarding the genetic architecture of the trait. To identify genomic regions associated with RT, we conducted a GWAS on SNPs in a cohort of 452 mid-first-lactation Canadian Holstein cows, followed by the annotation of genes and enrichment analyses of quantitative trait loci (QTL). Animals were genotyped using a medium-density SNP panel (50 K). Quality control measures were used to remove markers residing on nonautosomal chromosomes or with minor allele frequencies <5%, and SNP or animals with call rates lower than 90%. The SNP effects were estimated using single-step genomic BLUP. Significant markers were identified using a chromosome-wise modified Bonferroni correction, based on the expected number of independent chromosome segments. We identified 35 SNPs significantly associated with RT, mapping 34 genes within a 50-kbp interval up and downstream from these SNPs. Additionally, 19 QTL were found enriched in these genomic regions. Notably, genes such as ATP2B4, LDB3,WARS2, and PTPRO were identified, suggesting potential links to muscle fiber activity and milk solids percentage. The enriched QTL were associated with traits related to fat and protein synthesis and deposition in both milk and muscle tissues. Gene Ontology analysis highlighted terms related to muscle contraction and neuronal communication, consistent with the physiological processes underlying RT. Our findings offer new insights into the genetic architecture of RT, advancing the understanding of the physiological mechanisms governing this complex trait.