Climatic and glacio-eustatic information for the Late Paleozoic Ice Age is often extracted from cyclothems of the Midcontinent of North America. Although it is recognized that these important archives of global climate and environmental change are the result of waxing and waning of Gondwanan glaciers that produced widespread transgressive-regressive events, the relationships of conodont biofacies to changes in water depth, water chemistry, and other environmental parameters remain in dispute. Here, we investigate environmental gradients within the watermass of the Middle Pennsylvanian North American Midcontinent Sea (NAMS) based on geochemical proxies and conodont abundance patterns. To this purpose, we studied the Excello Shale at three geographically widely separated locations representing a ~500-km transect across the Midcontinent Shelf. We analyzed the organic (total organic carbon, carbon isotopes, and organic matter maturity) and inorganic geochemistry (major and trace elements) of the black and gray shale succession, as well as conodont abundance patterns and oxygen isotopic compositions, at 5–10 cm intervals through each study section.Our results suggest that the standard depth-stratification model of conodont biofacies in Pennsylvanian marine systems needs refinement. Although both Gondolella and Idioprioniodus are regarded as deep-water taxa, they differ in their stratigraphic distributions, with Idioprioniodus present through most of the Excello Shale but Gondolella confined to a narrow zone in the middle part. The Gondolella acme was not accompanied by pronounced changes in δ18O or trace-metal enrichment factors, implying that water depths and redox conditions were not the main controls on its distribution. Rather, the Gondolella acme is associated with the regressive condensation surface (RCS) of the core shale, which marks the onset of eustatic fall (renewed Gondwanan glaciation) and breakdown of the NAMS pycnocline. The latter process led to enhanced vertical overturn, mixing nutrients up into the ocean-surface layer and stimulating marine algal productivity, which was probably the trigger for blooms of Gondolella, suggesting that it was an opportunistic taxon. The upper part of the Excello is characterized by divergent δ18O compositions (~1‰ heavier in proximal areas), greater conodont abundance in offshore areas, and the appearance of Hindeodus exclusively in nearshore areas. This pattern suggests that Hindeodus was tolerant of somewhat more saline conditions, which developed during the climatic drying trend that accompanied the onset of the renewed Gondwanan glaciation. Our refined conodont biofacies model thus takes into consideration the effects of variable nutrient levels and watermass salinities in addition to water-depth controls.