AbstractPCIF1 (phosphorylated CTD interacting factor 1) is the first reported RNA N6,2′-O-dimethyladenosine (m⁶Am) methyltransferase. However, the pathological significance of PCIF1 and m⁶Am modification remains unknown. Here we find that both PCIF1 expression and m⁶Am modification are significantly elevated in gastric cancer tissues. Increased PCIF1 is associated with gastric cancer progression, and predicts poor prognosis. Silence of PCIF1 inhibits the proliferation and invasion of gastric cancer cells, and suppresses tumor growth and metastasis in mouse model. m⁶Am-seq analysis reveals TM9SF1 (transmembrane 9 superfamily member 1) as a target of PCIF1. PCIF1 modifies TM9SF1 mRNA with m⁶Am leading to decreased TM9SF1 translation. TM9SF1 reverses the effects of PCIF1 on gastric cancer cell aggressiveness. Collectively, our work uncovers an oncogenic function of PCIF1, providing insights into the critical role of m⁶Am modification in cancer progression.