Aflatoxin B(1) (AFB(1)) is a risk factor for hepatocellular carcinoma (HCC) in many low-resource countries. Although its metabolites bind at several positions in TP53, a mutation at codon 249 (AGG to AGT, arginine to serine, p.R249S) accounts for 90% of TP53 mutations in AFB(1)-related HCC. This specificity suggests that p.R249S confers a selective advantage during hepatocarcinogenesis. Using HCC cell lines, we show that p.R249S has lost the capacity to bind to p53 response elements and to transactivate p53 target genes. In p53-null Hep3B cells, stable transfection of p.R249S or of another mutant, p.R248Q, did not induce significant changes in cell proliferation and survival after cytotoxic stress. In contrast, in a cell line that constitutively expresses both p.R249S and the hepatitis B virus antigen HBx (PLC/PRF/5), silencing of either p.R249S or HBx by RNA interference slowed down proliferation, with no additive effects when both factors were silenced. Furthermore, the two proteins appear to form a complex. In human HCC samples, mutation at codon 249 did not correlate with p.R249S protein accumulation or HBx truncation status. We suggest that p.R249S may contribute to hepatocarcinogenesis through interaction with HBx, conferring a subtle growth advantage at early steps of the transformation process, but that this interaction is not required for progression to advanced HCC.