To determine the effect of supplemental feeding of Diamond V-XP yeast (XPY) alone or in combination with propionibacteria strain P169 on milk production, milk components, body weight, days to first and second ovulation, plasma insulin, and plasma and milk glucose, 31 primiparous and multiparous (MP) Holstein cows were fed one of three dietary treatments between 2 weeks prepartum to 30 weeks postpartum: (i) control (n = 10), fed a corn silage-based total mixed ration (TMR); (ii) XPY (n = 11), fed control TMR plus XPY (at 56 g/head/day); and (iii) P169+XPY (n = 10), received control TMR plus XPY plus P169 (at 6 x 10(11) cfu/head/day). After parturition, daily milk weights were recorded, and milk samples were collected twice weekly for milk component analyses. Daily uncorrected milk, solids-corrected milk, and 4% fat-corrected milk production for MP cows fed P169+XPY was 9-16% greater than control MP cows, but these increases were only evident during mid lactation (9-30 weeks). The percentage of milk fat was 8-18% greater in control than XPY and P169+XPY groups. Milk lactose percentage in MP cows fed P169+XPY was 3-5% greater than in control and XPY MP cows. Primiparous and MP cows fed P169+XPY had 28-32% greater milk glucose levels than control and XPY-fed cows. Diurnal plasma glucose concentration was not affected by diet in MP cows. Plasma insulin levels in MP cows fed P169+XPY were 30-34% greater than in other groups of MP cows. Milk glucose and plasma insulin responses to P169+XPY feeding suggest that P169+XPY might have enhanced gluconeogenesis and increased glucose uptake by the mammary gland in Holstein cows. Thus, a combined feed supplement of P169 and XPY may hold potential as a natural feed alternative to hormones and antibiotics to enhance lactational performance.