An experiment was conducted to evaluate the impact of feeding bio-fuel co-products on ruminal fermentation characteristics and composition of omasal digesta flow. Four ruminally cannulated Holstein steers (371 ± 5 kg) were used in a 4 × 4 Latin Square design. Omasal sample collection and triple marker technique was used to quantify fatty acid omasal flow. Treatments were applied as a 2 × 2 factorial where a steam flaked corn (SFC) basal diet (DGS-N CG-N) was replaced with 40% of diet DM as corn distillers grains (DGS; DGS-Y CG-N) or 10% of diet DM as crude glycerin (DGS-N CG-Y) or 40% of diet DM distillers grains and 10% of diet DM as crude glycerin (DGS-Y CG-Y). No effects were observed for the interaction of DGS and glycerin on measured rumen characteristics. Dietary inclusion of glycerin decreased (P = 0.05) ruminal content 4-h post feeding on a DM basis but did not influence DMI (P = 0.64). Feeding DGS had no effect (P = 0.34) on particulate passage to the omasum (kg/d) in spite of greater (P = 0.04) DMI. Feeding DGS reduced flow rate (% of rumen volume/h) (P = 0.05) but did not affect total VFA concentration (P = 0.46) or average ruminal pH (P = 0.72). No differences (P > 0.05) were observed in ruminal parameters when feeding glycerin, besides ruminal particulate content (kg) on DM basis (P = 0.05). An interaction of DGS and glycerin affected intake of stearic (P < 0.01), linoleic (P < 0.01), and linolenic acid (P < 0.01). An interaction of DGS and glycerin did not affect individual fatty acid flow with respect to intake for stearic (P = 0.17), linoleic (P = 0.18), or linolenic acid (P = 0.66). Dietary inclusion of glycerin had no impact on g of linolenic (P = 0.16) or linoleic (P = 0.32) acid transformed. A trend was identified for cattle fed diets with glycerin to have increased (P = 0.07) grams of conjugated linoleic acid (CLA; C18:2 cis-9, trans-11) per gram of linoleic acid intake, with no impact on the percent of saturated fat (P = 0.44) or unsaturated fat (P = 0.43) in omasal flow. For cattle fed diets with DGS, fewer grams of linoleic (P < 0.01) and linolenic (P < 0.01) were present in digesta flow per gram of intake. Inclusion of DGS in the treatment diets also increased (P < 0.01) stearic acid flow (g) and CLA flow (g) per gram of stearic and linoleic acid intake, respectively. Observed differences in CLA proportion post fermentation may indicate interrupted biohydrogenation when glycerin is fed.