Abstract Background The impact of microsatellite status on lymph node (LN) yield during lymphadenectomy and pathological examination has never been assessed in gastric cancer (GC). In this study, we aimed to appraise the association between microsatellite instability-high (MSI-H) and LN yield after curative gastrectomy. Methods We retrospectively analyzed 1757 patients with GC undergoing curative gastrectomy and divided them into two groups: MSI-H (n = 185(10.5%)) and microsatellite stability (MSS) (n = 1572(89.5%)), using a five-Bethesda-marker (NR-24, BAT-25, BAT-26, CAT-25, MONO-27) panel. The median LN count and the percentage of specimens with a minimum of 16 LNs (adequate LN ratio) were compared between the two groups. The log odds (LODDS) of positive LN count (PLNC) to negative LN count (NLNC) and the target LN examined threshold (TLNT(x%)) were calculated in both groups. Results Statistically significant differences were found in the median LN count between MSI-H and MSS groups for the complete cohort (30 vs. 28, p = 0.031), for patients undergoing distal gastrectomy (DG) (30 vs. 27, p = 0.002), for stage II patients undergoing DG (34 vs. 28, p = 0.005), and for LN-negative patients undergoing DG (28 vs. 24, p = 0.002). MSI-H was an independent factor for higher total LN count in patients undergoing DG (p = 0.011), but it was not statistically correlated to the adequate LN ratio. Statistically significant differences in PLNC, NLNC and LODDS were found between MSI-H GC and MSS GC (all p < 0.001). The TLNT(90%) for MSI-H and MSS groups were 31 and 25, respectively. TLNT(X%) of MSI-H GC was always higher than that of MSS GC regardless of the given value of X%. Conclusions MSI-H was associated with higher LN yield in patients undergoing gastrectomy for GC. Although MSI-H did not affect the adequacy of LN harvest, we speculate that a greater lymph node yield is required during pathological examination in MSI-H GC.