Objective Many rheumatoid arthritis (RA) patients have their disease controlled with anti-tumor necrosis factor alpha (anti-TNF) biologic therapies. However, a significant number of RA patients fail to respond to anti-TNF therapy. Here, we hypothesized that rare and low-frequency variants might influence anti-TNF treatment response. Methods We sequenced the coding region of 750 genes in 1,094 RA patients of European ancestry treated with anti-TNF medications. After quality control, we included 690 genes in the analysis. We applied single variants association and gene-based association tests to identify variants associated with anti-TNF treatment response. We also performed gene set analyses of 27 TNF pathway genes, considering TNF's key mechanistic role. Results We identified 14,420 functional variants, of which 6,934 were predicted as non-synonymous where 2,136 were further predicted to be damaging. Despite being a well-powered study, no single variant or gene showed study-wide significant association with change in disease activity outcome or EULAR response outcome. Intriguingly, we observed 3 genes, out of 27 with nominal signals of association (P<0.05), that were involved in the TNF signaling pathway. However, when we performed a rigorous gene set enrichment analysis based on association P values ranking, we observed no evidence of enrichment of association at genes involved in the TNF pathway (Penrichment =0.15, based on phenotype permutations). Conclusions Our study suggests that rare and low-frequency protein-coding variants in TNF signaling pathway genes or other genes do not contribute substantially to anti-TNF treatment response in RA patients. This article is protected by copyright. All rights reserved.