Background: In lung cancer, miR-29b can act as a tumor suppressor, influencing epigenetic regulation, cell proliferation, differentiation, apoptosis, metastasis, and chemosensitivity. However, its expression and biological function in Xuanwei lung cancer (XWLC) remains unknown. Methods: Fifty pairs of XWLC and matched normal tissues were collected and analyzed for the expression of miR-29b by quantitative RT-PCR. MiR-29b precursor and inhibitor were transfected into XWLC-05 cells to investigate its role on regulating cell proliferation by MTT assay and cell apoptosis by caspase 3/7/9 assay in vitro. The target genes of miR-29b were predicted and determined by bioinformatics analysis and luciferase reporter assay. Results: qRT-PCR results showed that the expression level of miR-29b in XWLC tissues was significantly lower than that in the matched normal tissues, with a proportion of 86%. The low expression of miR-29b was significantly associated with lymphatic metastasis. MiR-29b could suppress cell proliferation and promote apoptosis in XWLC-05 cells. According to the bioinformatics analysis and luciferase reporter assay results, PUMA was a target gene of miR-29b in XWLC. Conclusions: Our studies provided evidence for the involvement of miR-29b in XWLC lymphatic metastasis and suggested that miR-29b may serve as a new biomarker for diagnosis of XWLC or therapeutic targets.