Horizontal transfer of plasmids by conjugation is a fundamental mechanism driving the widespread dissemination of drug resistance among bacterial populations. The successful colonization of a new host cell necessitates the plasmid to navigate through a series of sequential steps, each dependent on specific plasmid or host factors. This review explores recent advancements in comprehending the cellular and molecular mechanisms that govern plasmid transmission, establishment, and long-term maintenance. Adopting a plasmid-centric perspective, we describe the critical steps and bottlenecks in the plasmid’s journey toward a new host cell, encompassing exploration and contact initiation, invasion, establishment and control, and assimilation.