C-reactive protein (CRP), an acute phase protein in humans, is predominantly produced by hepatocytes in response to interleukin-6 (IL-6). Several epidemiological studies have reported that dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) is inversely associated with serum CRP concentration. However, the molecular mechanism by which n-3 PUFAs reduce the serum CRP level in HepG2 cells remains unclear. The aims of this study were to examine the effect of the n-3 PUFAs, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), on the modulation of IL-6-induced CRP expression and to explore its possible mechanisms. We demonstrated that DHA and EPA inhibited IL-6-induced CRP protein and mRNA expression, as well as reduced CRP promoter activity in HepG2 cells. Knockdown of Signal Transducer and Activator of Transcription 3 (STAT3) and CCAAT box/Enhancer-Binding Protein β (C/EBPβ) by small interfering RNAs (siRNAs) significantly decreased IL-6-induced CRP promoter activity. Gel electrophoresis mobility shift assays (EMSA) showed that pretreatment with DHA and EPA decreased IL-6-induced STAT3 DNA binding activity but not C/EBPβ. By western blot analysis, DHA and EPA inhibited IL-6-induced STAT3 phosphorylation but not ERK1/2 or C/EBPβ. The suppression of the phosphorylation of STAT3 by DHA and EPA was further verified by immunofluorescence staining. Taken together, our results demonstrate that DHA and EPA are able to reduce IL-6-induced CRP expression in HepG2 cells via an inhibition of STAT3 activation. This mechanism, which explains the inhibitory effect of n-3 PUFAs on the CRP expression, provides new insights into the beneficial anti-inflammatory effect of n-3 PUFAs.