This study aimed to explore novel microRNAs in plasma for predicting chemoresistance in adjuvant chemotherapy for patients with gastric cancer (GC). We used the Toray 3D-Gene microRNA arraybased approach to compare preoperative plasma microRNA levels between GC patients with and without recurrences after curative gastrectomy. All patients underwent adjuvant chemotherapy with S-1, an oral fluoropyrimidine. Of 2566 candidates, six candidate microRNAs (miR-1229-3p, 1249-5p, 762, 711, 1268a and 1260b), which were highly expressed in the preoperative plasma of patients with subsequent recurrences, were selected. In a large-scale validation analysis by quantitative RT-PCR, we focused on high plasma levels of miR-1229-3p, which was an independent poor prognostic factor for recurrence free survival (P = 0.009, HR = 3.71). Overexpression of miR-1229-3p in GC cells induced significant chemoresistance to 5-fluorouracil (5-FU), up-regulation of thymidylate synthase (TS) and dihydroprimidine dehydrogenase (DPD) and down-regulation of SLC22A7 both in vitro and in vivo. Intraperitoneal injection of miR-1229-3p in mice induced significant chemoresistance to 5-FU, accompanied by high levels of miR-1229-3p in plasma and tumor tissue. These findings suggest that plasma miR-1229-3p might be a clinically useful biomarker for predicting chemoresistance to S-1 and selecting other or combined intensive chemotherapy regimens in GC patients.Gastric cancer (GC) is the third leading cause of cancer-related death both in Japan and worldwide. Although the surgical techniques, perioperative chemotherapy regimens and perioperative management have greatly improved, GC remains one of the most common cancer types and constitutes a global health problem 1,2 . Curative gastrectomy with lymphadenectomy is recognized as an opportunity for macroscopic tumor clearance and a cure for GC. However, the effect of surgical resection is restricted to local control of the primary tumor 3,4 ; thereby, it cannot prevent recurrence due to micrometastasis. Therefore, perioperative chemotherapy has been recommended to achieve microscopic tumor clearance in advanced GC. In Japan, an adjuvant chemotherapy regimen with S-1, an oral fluoropyrimidine, following curative gastrectomy for Stage II or III GC is recommended as a standard treatment to improve the survival rate of GC patients, based on the results of the ACTS-GC (Adjuvant Chemotherapy Trial of TS-1 for Gastric Cancer) 5,6 . However, some GC patients after curative gastrectomy followed by adjuvant S-1 chemotherapy had recurrences 7 . Nevertheless, no companion diagnostic marker has been detected for predicting chemoresistance to 5-fluorouracil (5-FU) or the oral modulated 5-FU prodrug S-1 in GC.MicroRNAs (miRNAs), which are small noncoding RNAs, modulate the translation of specific protein-coding genes. miRNA was discovered in 1993, and a lot of studies have identified alterations in miRNA expression that contribute to the progression of various diseases, including the progression and development of several can...