BACKGROUND: The role of first-line single-agent rituximab immunotherapy in follicular lymphoma (FL) remains debated, as most patients eventually undergo chemotherapy. METHODS: In this study, we retrospectively analyzed 81 FL patients treated with first-line single-agent rituximab monotherapy with ( RESULTS: After a median follow-up of 11 years, overall survival (OS) and progression-free survival (PFS) rates were 85% and 32%, respectively. Targeted gene expression profiling (T-GEP) was performed in 40 patients, revealing a 26-gene expression signature distinguishing complete responders and non-responders. This signature included genes involved in T-regulatory (Treg) and natural-killer cell activity, and interleukin-17 signaling. A simplified 14-gene prognostic score (ImSig) enabled accurate outcome stratification in terms of PFS. These data were validated in silico using two independent publicly available cohorts of FL patients treated with chemoimmunotherapy. Deconvolution analyses demonstrated an enrichment in Treg cells in high-risk ImSig patients, which was validated by immunohistochemistry. CONCLUSIONS: These findings demonstrate that the efficacy of front-line anti-CD20 immunotherapy may depend on microenvironment-related factors, and that specific immune signatures could identify patient subsets obtaining long-term benefit from a chemo-free immunotherapeutic approach. TRIAL REGISTRATION: The authors have confirmed clinical trial registration is not needed for this submission.