Average genome size is an important, yet often overlooked, property of microbial communities. We developed MicrobeCensus to rapidly and accurately estimate average genome size from shotgun metagenomic data and applied our tool to 1,352 human microbiome samples. We found that average genome size differs significantly within and between body sites and tracks with major functional and taxonomic differences. In the gut, average genome size is positively correlated with the abundance of Bacteroides and genes related to carbohydrate metabolism. Importantly, we found that average genome size variation can bias comparative analyses, and that normalization improves detection of differentially abundant genes.