Emerging evidence has shown that microRNAs (miRNAs) play essential roles in regulating human cancers development and progression. However, the underlying mechanisms remain to be further explored. MiRNAs are a class of endogenous, non-coding, 18-24 nucleotide length single-strand RNAs that moderate gene expression primarily at post-transcriptional level. There is a growing body of literature that recognizes the importance of microRNA (miR)-129 during the development of cancers. Aberrant expression of miR-129 has been detected in various types of human cancers and the validated target genes are involved in cancer-related biological processes such as DNA methylation, cell proliferation, apoptosis, cell cycle, and metastasis. In this review, we summarized the roles of miR-129 family members and their target genes in tumorigenesis and clinical treatment of human cancers, highlighting the potential roles of miR-129 as biomarkers for cancer diagnosis and prognosis, and promising tools for cancer treatment.