Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, and it is characterized by a high incidence. It is important to understand the molecular mechanisms that determine the progression and metastasis of NSCLC in order to develop more effective therapies and identify novel diagnostic indicators of NSCLC. RSPH14 has been reported to be related to multiple human diseases, including duodenal adenocarcinoma and meningiomas, but the role of RSPH14 in NSCLC remains unclear. The present study aimed to investigate the molecular function and clinical significance of RSPH14 in NSCLC. Analyses of public datasets and clinical samples demonstrated that RSPH14 expression was upregulated in NSCLC samples compared with normal samples. In addition, high RSPH14 expression was associated with a shorter overall survival time in patients with NSCLC. Notably, RSPH14 knockdown suppressed the proliferation and cell cycle progression and enhanced the apoptosis of NSCLC cells. Mechanically, tandem mass tag analysis demonstrated that RSPH14 can affect multiple processes, including the AMPK signaling pathway, calcium ion import regulation, glucose transmembrane transporter activity, and glucose transmembrane transport. Taken together, the results of the present study suggest that RSPH14 may be a promising prognostic factor and therapeutic target for NSCLC.