This study aimed to investigate the expression and cellular function of the centromeric family of proteins (CENPs), especially centromere protein I (CENP-I), in gastric cancer (GC) and identified its clinical significance and cellular functions. CENP-I expression in GC was studied by cDNA microarray, quantitative real-time PCR (qRT-PCR), and immunohistochemistry (IHC), and using datasets from The Cancer Genome Atlas (TCGA), UALCAN, and Gene Expression Omnibus (GEO) databases. Microarray and bioinformatic analyses identified upregulated CENP-A/E/F/H/I/K/P/W and HJURP in stomach adenocarcinoma (STAD), but not in signet ring cell carcinoma (SRCC). Significantly higher CENP-I mRNA expression was also confirmed in 40 pairs of GC tissues than in paired normal gastric tissues by qRT-PCR (P<.001). IHC showed that elevated CENP-I expression was associated with higher tumor stage, lymph node invasion, increased HER2-positive rate (36.7% vs 10.0%), and intestinal Lauren classification in 69 GC samples compared to paired paracancerous normal tissues. The survival of the high-CENP-I group members was poor compared with that of the low-CENP-I group (P = .0011). Cox univariate regression analysis identified tumor size (P = .008), HER2 status (P = .027), and CENP-I expression (P = .049) were independent prognostic factors of GC. The cellular function of CENP-I was studied in MKN45 and MKN28 GC cell lines in vitro. Cell proliferation, migration, and apoptosis were determined using CCK-8, transwell assay, TUNEL assay, and flow cytometry. Our results showed that CENP-I promoted GC cell proliferation, inhibited apoptosis, facilitated cell migration, and induced epithelial-mesenchymal transition (EMT), possibly by activating the AKT pathway. CENP-I expression was correlated with genetic signatures of the proliferative subtype of GC, characterized by intestinal Lauren classification, HER2 amplification, and TP53 mutation. In conclusion, this study revealed an elevated CENP-I expression in GC, which was associated with malignant features and poor prognosis of GC patients, and identified its function in modulating cell proliferation, apoptosis, and migration.Keywords: Lauren classification; centromere protein I; gastric cancer; mitotic checkpoints; proliferative subtype.