MiRNA-375 has been reported to play critical roles in a variety of cancers. To unravel its biological roles, especially its specific mechanisms of action in lung squamous cell carcinoma (LUSC), LUSC tissue microarrays and miRNAscope were performed to identify the miR-375 expression. Associations with clinicopathologic features, survival, and the prognostic value of miR-375 in LUSC were clarified in a retrospective study of 90 pairs of LUSC tissues. In vitro and in vivo gain- and loss-of-function assays were conducted to validate the effects and mechanism of miR-375 in LUSC. The mechanism responsible for interactions was verified by dual-luciferase reporter gene assay, immunoprecipitation (IP) analysis, immunofluorescence (IF) assay and ubiquitination assay. We found that miR-375 had higher expression in noncancerous adjacent tissues than in LUSC tissues. Clinicopathologic analyses showed that miR-375 expression was correlated with pathologic stage and was an independent predictor of overall survival (OS) for LUSC. MiR-375, as a tumor inhibitor, inhibited proliferation and metastasis while promoting apoptosis of LUSC cells. Mechanistic research indicated that miR-375 targeted ubiquitin-protein ligase E3A (UBE3A), which in turn promoted the activity of the ERK signaling pathway via ubiquitin-mediated dual-specificity protein phosphatase 1 (DUSP1) degradation. Collectively, we propose a novel mechanism of tumorigenesis and metastasis of LUSC via the miR-375/UBE3A/DUSP1/ERK axis, which could potentially facilitate new strategies for the treatment of LUSC.