The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. Great attention has been put on improved secretory production of active pharmaceutical ingredients, and the secretory pathway of this eukaryotic host has been the playground of diverse strain engineering studies, aiming at enhanced cellular capacities for folding and trafficking of the target proteins. However, the cellular quality assessment for secretory proteins remains mostly unpredictable, and different target proteins often do not picture similar secretion yields, underlining the dependency of efficient secretion on the physicochemical properties of the protein of interest. In this study, two human insulin analog precursors (IAPs) with minor differences in their amino acid sequences were used as model secretory proteins. No differences between cells expressing these two proteins were found in the IAP transcript levels, gene copy numbers, or intra-cellularly accumulated proteins, yet a more than sevenfold difference in their secretion yields was found. Physiological characterization of cells expressing these proteins in batch processes revealed no significant difference in their specific growth rate, but an altered overflow metabolism. Global transcriptome analysis carried out in chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated and indicated an increased degradation of the IAP with the low secretion yield. In silico protein structure modeling of the IAPs suggested a difference in conformational stability, induced by the amino acid substitution, which most likely resulted in disparity in trafficking through the secretory pathway and thus a large difference in secretion yields. Biotechnol. Bioeng. 2013;110: 2764–2774. © 2013 Wiley Periodicals, Inc.