Cotyledons are an important source of nutrients and food reserves for young soybean plants before they become autotrophic. However, the importance of the cotyledons becomes unclear once the unifoliolate leaves have unrolled and the plants are in the cotyledon growth stage (VC). Field experiments conducted in 2005 at DeKalb and Urbana, Illinois included removal of one cotyledon, both cotyledons, both unifoliolate leaves, or one cotyledon plus both unifoliolate leaves from all plants, removal of both cotyledons from every other plant, and a non-defoliated control. Across defoliation levels, grain yield and seed weight at DeKalb were 20 and 46% greater than those at Urbana, respectively, indicating more favorable growing conditions during the seed filling period at DeKalb. Grain yield, seed weight, and final plant height were not affected by any of the treatments at DeKalb, while seed weight was not affected by defoliation at Urbana. On average, removal of the unifoliolate leaves or one cotyledon plus the unifoliolate leaves at Urbana reduced grain yield by 13% and final plant height by 11% when compared to the control. Removal of one or both cotyledons on all plants, or both cotyledons from every other plant at Urbana did not reduce grain yield or final plant height. These results indicate that loss of cotyledons when soybean is in the VC growth stage may be of little importance, while loss of the unifoliolate leaves, resulting in regrowth from the cotyledonary buds, can significantly reduce yield and height, especially when less than ideal growing conditions occur during seed fill.