Podocin (NPHS2) is a component of the glomerular slit-diaphragm, with major regulatory functions in renal permeability of proteins. Loss of podocin and decrease in resynthesis may influence the outcome of proteinuric renal disease such as segmental glomerulosclerosis (FSGS), and promoter functionality plays a key role in this process. NPHS2 promoter variants with functional activity may be a part of the problem of podocin resynthesis. We sequenced NPHS2 promoter region from -628 to ATG in a large cohort of 260 nephrotic patients (161 with FSGS) who were presenting proteinuria from moderate to severe and were receiving or had received modular therapies according to their sensitivity to steroids and other immune modulators. Three sequence variants (-236C>T, -52C>G, -26C>G) were identified in our study population that gave an allele frequency below 1% (5 patients out of 520 alleles). Functional implications were shown for each variants that were most evident for -52C>G and -26C>G (-50% of luciferase expression compared to the wild-type sequence, p < 0.01). Consensus analysis for homology of the -52 region with regulatory factors revealed homology for USF1 and the sum of experiments with gel retardation and with cells silenced for USF1 confirmed that this factor regulates NPHS2 expression at this site. In conclusion, three functional variants in NPHS2 promoter have been identified in a large cohort of patients with nephrotic syndrome and FSGS that have a frequency <1%. One of these (i.e., -52C>G) is associated with a poor clinical outcome and evolution to end-stage renal failure. USF1 was identified as the transcriptional factor regulating NPHS2 at this site. Even if not sufficient to cause FSGS per se, these variants could represent modifiers for severity and/or progression of the disease.