Circulating n-3 polyunsaturated fatty acids (n-3 PUFAs), which integrate endogenous and exogenous n-3 PUFAs, can be better used to investigate the relationship between n-3 PUFAs and disease. However, studies examining the associations between circulating n-3 PUFAs and colorectal cancer (CRC) risk were limited and the results remained inconclusive. This case-control study aimed to examine the association between serum n-3 PUFAs and CRC risk in Chinese population. A total of 680 CRC cases and 680 sex and age-matched (5-year interval) controls were included. Fatty acids were assayed by gas chromatography. Odds ratios (ORs) and 95% confidence interval (CI) were calculated using multivariable logistic regression after adjustment for potential confounders. Higher level of serum α-linolenic acid (ALA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), long-chain n-3 PUFAs, and total n-3 PUFAs were associated with lower odds of CRC. The adjusted ORs and 95% CI were 0.34 (0.24-0.49, Ptrend< 0.001) for ALA, 0.57 (0.40-0.80, Ptrend<0.001) for DPA, 0.48 (0.34-0.68, Ptrend< 0.001) for DHA, 0.39 (0.27-0.56, Ptrend< 0.001) for long-chain n-3 PUFAs, and 0.31 (0.22-0.45, Ptrend< 0.001) for total n-3 PUFAs comparing the highest with the lowest quartile. However, there was no statistically significant association between eicosapentaenoic acid (EPA) and odds of CRC. Analysis stratified by sex showed that ALA, DHA, long-chain n-3 PUFAs and total n-3 PUFAs were inversely associated with odds of CRC in both sexes. This study indicated that serum ALA, DPA, DHA, long-chain n-3 PUFAs and total n-3 PUFAs was inversely associated with odds of having CRC in Chinese population.