A comprehensive understanding of genotype-phenotype links in bacteria is the primary theme of bacterial functional genomics. Transposon sequencing (Tn-seq) or its equivalent methods that combine random transposon mutagenesis and next-generation sequencing (NGS) represent a powerful approach to understand gene functions in bacteria on a genome-wide scale. This approach has been utilized in a variety of bacterial species to provide comprehensive information on gene functions related to various phenotypes or biological processes of significance. With further improvements in the molecular protocol for specific amplification of transposon junction sequences and increasing capacity of next generation sequencing technologies, the applications of Tn-seq have been expanding to tackle questions that are important yet difficult to address in the past. In this review, we will discuss the technical aspects of different Tn-seq methods along with their pros and cons to provide a helpful guidance for those who want to implement or improve Tn-seq for their own research projects. In addition, we also provide a comprehensive summary of recent published studies based on Tn-seq methods to give an updated perspective on the current and emerging applications of Tn-seq.