PURPOSE: Small extracellular vesicles (sEV) containing proteins and RNAs play important roles as intercellular signal mediators. A critical issue is that there are multiple methods to prepare sEV fractions. The purpose of this study was to examine whether cancer cell-derived sEV fractions prepared by different isolation methods show similar responses for the induction of inflammatory cytokines in macrophages. METHODS: sEV fractions from the conditioned medium of MCF-7 cells were prepared by ultracentrifugation (UC), the MagCapture Exosome Isolation Kit PS (PS), or the ExoQuick-TC kit (EQ). The mRNA levels of inflammatory cytokines in differentiated THP-1 cells treated with the sEV fractions were evaluated. RESULTS: The yields of sEV fractions obtained from 1 mL conditioned medium by UC, PS, or EQ were 3.2×10 particles (0.27 μg protein), 12.8×10 particles (0.87 μg protein) and 23.5 ×10 particles (4.50 μg protein), respectively. The average particle sizes in the UC, PS, and EQ fractions were 184.8 ± 1.8 nm, 157.8 ± 1.3 nm and 165.8 ± 1.1 nm, respectively. CD9 and CD81, markers of sEV, were most highly detected in the PS fraction, followed by the EQ and UC fractions. These results suggest that PS gave sEV with relatively high purity, and many protein contaminants appear to be included in the EQ fraction. The mRNA levels of inflammatory cytokines in THP-1 macrophages were most prominently increased by treatment with the UC fraction, followed by the EQ and PS fractions, suggesting that contaminants rather than sEV may largely induce an inflammatory response. CONCLUSION: The isolation method affects the evaluation of sEV function.