Abstract YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with 15 N/ 14 N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.