Laryngeal squamous cell carcinoma (LSCC) is one of the most frequently diagnosed head and neck cancers worldwide. Increasing evidence suggests that microRNAs (miRNAs/miRs) regulate the progression of tumorigenesis and the malignant behaviors of cancer cells. The aim of this study was to investigate the function and underlying mechanism of miR-375-3p in LSCC. The expression of miR-375-3p in LSCC tissues and cells was detected using reverse transcription-quantitative PCR. The effects of miR-375-3p on the malignant phenotype of LSCC cells was determined using the Cell Counting Kit-8 assay and flow cytometry. The targets of miR-375-3p were predicted using the miRDB database and confirmed by the luciferase reporter assay. The results of the present study demonstrated that miR-375-3p was downregulated in LSCC tissues and cell lines. Furthermore, overexpression of miR-375-3p significantly suppressed the proliferation and cell cycle progression of LSCC cells. Overexpression of miR-375-3p also increased LSCC cell apoptosis. Mechanistical analysis indicated that miR-375-3p bound the 3'-untranslated region of the hepatocyte nuclear factor 1β (HNF1β) and decreased its expression in LSCC cells. Consistent with the role of HNF1β in glucose metabolism, overexpression of miR-375-3p significantly inhibited glucose consumption and lactate production in LSCC cells. Transfection with HNF1β notably reversed the inhibitory effect of miR-375-3p on the proliferation of LSCC cells. Collectively, these results indicate the tumor suppressive role of miR-375-3p in LSCC via HNF1β, suggesting that miR-375-3p may serve as a potential target in the treatment of LSCC.