The transcriptome and proteome of Arabidopsis thaliana are reduced in nitrogen content when compared with other taxa, which may result from ecological nitrogen limitation. We hypothesized that if the A. thaliana transcriptome is selected for a low nitrogen content, nitrogen-reducing derived alleles of single nucleotide polymorphisms (SNPs) should segregate at higher frequencies than nitrogen-increasing alleles. This pattern should be stronger in populations with a larger effective population size (Ne) if natural selection is more efficient in large than in small populations. We analyzed variation in the nitrogen content in the transcriptome of 80 natural accessions of A. thaliana. In contrast to our expectations, derived alleles increase the nitrogen content in all accessions, and there is a positive correlation between nitrogen difference and derived allele frequency, which is strongest with nonsynonymous SNPs (nsSNPs). Also, there is a positive correlation between nitrogen difference and Ne that was mainly caused by nsSNPs. These observations led us to reject the hypothesis that the transcriptome of A. thaliana is currently under selection to reduce nitrogen content. Instead, we show that a change in nitrogen content is a side effect of interacting evolutionary factors that influence base composition and include mutational bias, purifying selection of functionally deleterious alleles, and GC-biased gene conversion. We provide strong evidence that GC-biased gene conversion may play an important role for base composition in the highly selfing plant A. thaliana.