About 30% of proteins synthesized in bacteria perform their functions outside of the cytoplasm and have to be inserted into or translocated across the cytoplasmic membrane. The primary system for protein translocation is the Secretory (Sec) pathway. Its essential components include the membrane-embedded protein-conducting channel SecYEG, the motor ATPase SecA, and the YidC insertase, and a number of accessory integral and peripheral membrane proteins, which facilitate targeting and translocation. Structural and in vitro functional studies on the Sec pathway have been carried out either in detergents or in model membranes, such as lipid monolayers, supported lipid bilayers, and (proteo-)liposomes. However, detergents may alter structural and functional properties of studied proteins, while the sample heterogeneity and protein aggregation occurring in large-scale model membranes often interfere with experimental analysis. Here, we review a recent progress in isolating Sec components within lipid-based particles, nanodiscs, for biophysical, biochemical, and structural analysis. Nanodiscs have been successfully applied to investigate oligomeric states of individual Sec components, to monitor structural dynamics of proteins and their assembly into functional complexes, and to reconstitute translocation and membrane insertion reactions. Cryo-electron microscopy of nanodisc-reconstituted SecYEG and YidC in complex with ribosomes visualized intermediates on membrane protein insertion and demonstrated structural dynamics of insertases. Nanodisc-based experiments have highlighted the importance of the physiologically relevant molecular environment for functionality of membrane-embedded components, but also for membrane-associated targeting machinery, and suggested nanodiscs as a powerful platform for further studies, including high-resolution structural analysis.