Maize (Zea mays L.) is a major dietary source of human caloric intake. Grain filling, the developmental stage of the seed during which starch and proteins accumulate, is of great interest in plant biology and agronomy. However, proteomic datasets covering maize seed development, especially during grain filling, are much scarcer than transcriptomic datasets, largely due to the labor-intensive and costly nature of the large-scale analysis required for proteomics. Here, we searched for proteins that showed changes in abundance during four time-points covering the middle stages of grain filling by two-dimensional electrophoresis, MALDI-TOF, and database searches. We detected 1384 protein spots, of which 48 exhibited differential accumulation during grain filling. Of those, we identified the underlying protein for 32 spots: they included enzymes of carbohydrate metabolism, stress-related proteins, and storage proteins, the latter of which represented 34% of all changing proteins during grain filling. Proteins related to carbohydrate metabolism reached their maximum accumulation around 15–20 days after pollination (DAP) and subsequently dropped until 30 DAP. The rise of stress-related proteins such as heat shock proteins demonstrated their involvement in grain filling and seed maturation. This study catalogues the proteome changes during grain filling and provides basic but critical information regarding the biological changes during maize kernel development.