As a potential oncogene, nucleolar and spindle‑associated protein 1 (NUSAP1) is involved in the regulation of tumor cell proliferation, metastasis and drug resistance. However, the role of NUSAP1 in non‑small cell lung cancer (NSCLC) remains unclear. The present study aimed to investigate the biological function and underlying molecular mechanisms of NUSAP1 in NSCLC. NUSAP1 expression was measured in NSCLC tissues and cell lines via immunohistochemistry and western blotting, respectively. NSCLC cell lines stably inhibiting NUSAP1 were established to investigate its effects on cell proliferation, colony formation and invasion, and on in vivo tumorigenicity. Additionally, the upstream and downstream mechanisms of NUSAP1 in regulating NSCLC progression were investigated. The results indicated that NUSAP1 expression was upregulated in NSCLC tissues and cell lines. High NUSAP1 expression was associated with tumor size, TNM stage, lymph node metastasis and poor patient survival, whereas knockdown of NUSAP1 inhibited NSCLC cell proliferation, colony formation and invasion. Furthermore, downregulation of NUSAP1 decreased the growth of NSCLC xenografts in vivo. In addition, myocyte enhancer factor 2D (MEF2D) directly targeted the NUSAP1 promoter, thereby enhancing the mRNA and protein expression levels of NUSAP1. Moreover, the results demonstrated that MEF2D expression was upregulated in NSCLC tissues and was positively correlated with NUSAP1 expression. MEF2D‑knockdown decreased NSCLC cell proliferation, colony formation and invasion. NUSAP1 upregulation reversed the effects of MEF2D‑knockdown on NSCLC progression. Furthermore, it was observed that MEF2D‑knockdown inhibited the accumulation and nuclear translocation of β‑catenin, thereby repressing the activation of the Wnt/β‑catenin signaling pathway in NSCLC cells, whereas NUSAP1 upregulation rescued the effects of MEF2D‑knockdown on the activation of the Wnt/β‑catenin signaling pathway. In conclusion, the findings of the present study indicated that the MEF2D/NUSAP1 signaling pathway promoted NSCLC progression by inducing the activation of Wnt/β‑catenin signaling, and this novel mechanism may represent a potential treatment target for patients with NSCLC.