In some studies, the prognostic and predictive significance of M30 and M65 has been reported to detect response to chemotherapy. In the present study, we aimed at determining the changes of serum M30 and M65 values after chemotherapy and the impact of these values on treatment response and progression-free survival (PFS) and overall survival (OS) of patients with advanced gastric cancer. A total of 31 patients with advanced gastric cancer was included. M30 and M65 values were measured by a quantitative enzyme-linked immunosorbent assay (ELISA) method in serum samples before and 48 h after the first chemotherapy cycle. Pre- and postchemotherapy values of M30 and M65 were compared. The difference between the mean values of serum M30 and M65 before and after chemotherapy was calculated and the prognostic significance of changes for survival was evaluated by univariate and multivariate analysis. Logistic regression analysis was performed to predict response to chemotherapy. Serum M30 and M65 levels were found to be increased significantly after chemotherapy (M30, 582.7 ± 111.5 U/l [pre mean] vs. 983.3 ± 214.1 U/l [post mean], p = 0.01; M65, 2,061.7 ± 431.2 U/l [pre mean] vs. 2,646.3 ± 433.1 U/l [post mean], p = 0.003). Means of the differences of M30 and M65 levels before and 48 h after chemotherapy were 400.5 ± 190 U/l ([M30-difference] M30-D) and 584.6 ± 335.4 U/l (M65-D), respectively. Patients with serum M30-D of <400.5 U/l had better median PFS and OS times than patients with M30-D >400.5 U/l (PFS, 9.9 vs. 4.3 months, p = 0.018 and OS, 13.6 vs. 8.1 months, p = 0.029). In addition, median PFS and OS intervals in patients with serum M65-D > 584.6 U/l were significantly worse than those of patients whose M65-D was lower than or equal to 584.6 U/l (4.1 vs. 11.4 months for PFS, p = 0.002 and 5.7 vs. 13.6 months for OS, p = 0.005). Patients with values above M30-D and M65-D had a better tumor response compared with patients with values below M30-D and M65-D (p = 0.02 and p = 0.006, respectively). In the logistic regression analysis, only M65-D was significantly found to be an independent factor in predicting response to chemotherapy (p = 0.018, OR:1.4). However, only M30 levels after chemotherapy were found to be an independent prognostic factor for PFS in the multivariate analysis. These results showed for the first time that both M30 and M65 in serum samples of patients with advanced gastric cancer were elevated 48 h after chemotherapy and these were poor prognostic factors for both PFS and OS of patients. Moreover, increased serum M65 levels after chemotherapy can be predict tumor response.