The objective of this study was to evaluate the effect of seed size, protein content and N application timing on barley seedling vigor and grain yield. Two seed sizes, three protein levels and two genotypes (Hazen and Hazen semi-dwarf, SD, isotype pair) were used in a laboratory and in a field study. In a greenhouse study with Hazen only, N application timing (at sowing and 20 days after emergence) treatments were added to the other treatments. High seed protein content and large seed positively affected coleoptile length, root dry weight and seedling dry weight at early stages of plant development, but had no effect on emergence rate index and plant and root dry weights at 40 days after emergence. Both the standard height and semi-dwarf isotypes responded similarly. The delay of N supply until 20 days after emergence negatively affected the initial seedling growth, but had no effect on plant dry weight 40 days after emergence in the greenhouse. Grain yield and yield components were not affected by seed protein content nor seed size under field conditions.