Objective The aim of this study was to investigate the expression of miR-320a and cephalospermoma syndrome protein(CYLD)in patients with gastric cancer and its relationship with clinicopathological characteristics and prognosis. Methods A total of 460 patients with gastric cancer underwent tumor resection in our hospital from March 2013 to November 2014 were enrolled. Tumor tissues,non-tumor gastric mucosa tissues and normal tissues were collected. The expression of miR-320a and CYLD at levels of mRNA and protein were detected by Real-Time PCR,immunohistochemistry and Western blotting. The relationship between the expression of miR-320a and CYLD,and the clinicopathological features&prognosis of gastric cancer patients was analyzed. Results The relative expression of miR-320a and CYLD at the mRNA level in tumor tissues was(0. 37 ± 0. 09),(0. 91 ± 0. 23),and the relative expression in non-tumor tissues was(0. 86 ± 0. 15),(1. 56 ± 0. 42),respectively. The relative expression of miR-320a and CYLD mRNA in tumor tissues was significantly different from non-tumor tissues(t=60. 078,29. 113,P=0. 000),the positive ex-pression rate of CYLD protein in tumor tissues was 43. 48% when compared to 73. 91% in non-tumor tissues. The difference was statistically significant(χ2=86. 624,P=0. 003). The expression level of miR-320a was significantly associated with the diameter of the tumor and lymph node metastasis(χ2=25. 859 and 13. 742,P<0. 05). The expression of CYLD was also significantly associated with the TNM stage and degree of tumor differentiation(χ2=37. 725 and 59. 323,P<0. 05). The median survival of patients with low miR-320a expression(20. 36 months,95% CI:19. 252 ~21. 462 months) and those with high miR -320a expression(28. 29 months,95% CI:27.158~29.412 months)were statistically significant(χ2=87.967,P<0.001).The median survival of patients with CYLD negative expression(17. 70 months,95% CI:16. 599~18. 796 months)and those with CYLD positive expression(26. 74 months,95% CI:25. 474~27. 997 months)were statistically significant(χ2=109. 887,P<0. 001);The median survival of patients with the co-expressed miR-320a and CYLD was(29. 01 months,95% CI:26. 831~28. 946 months)and those with the non-co-expressed miR-320a and CYLD(17. 13 months,95% CI:17. 214~19. 568 months)were statistically significant(χ2=117. 680,P<0. 001). There showed a positive correlation between the expression of miR-320a and CYLD at mRNA level(r=0. 607,P<0. 001);miR-320a at the low expression,CYLD at the negative expression,TNM staging,lymph node metastasis and degree of tumor differen-tiation were independent risk factors for the prognosis of gastric cancer(HR=1. 939,2. 180,1. 561,1. 719,1. 608,95% CI:1. 141~3.295,1.252~3.796,1.014~2.403,1.115~2.650,1.097~2.357,respectively)(P<0.05).Conclusion The expressions of miR-320a and CYLD in tumor tissues of patients with gastric cancer is significantly decreased,which is related to the occurrence and development of diseases,and poor prognosis. It is a potential target for diagnosis and treatment of gastric cancer.