Renal clear cell carcinoma (RCC) is the most common pathological type of renal carcinoma and drug resistance often occurs. We studied the effect of hsa_circ_0035483 on gemcitabine sensitivity in RCC, and explored its regulatory effect on downstream hsa-miR-335 and Cyclin B1 (CCNB1). High-throughput sequencing was used to analyze the differentially expressed circRNA in RCC. The expressions of hsa_circ_0035483, hsa-miR-335, CCNB1, and autophagy-related proteins were detected by RT-PCR or Western blot. The target relationships were revealed by RNA pulldown assay and dual luciferase report assay. Autophagy marker LC3 was detected by immunofluorescence. Cell viability was detected by MTT assay. Hsa_circ_0035483 can facilitate gemcitabine-induced autophagy, and enhance the resistance of RCC to gemcitabine. Hsa-miR-335 is the target regulatory point of hsa_circ_0035483. In addition, hsa_circ_0035483 promotes autophagy and tumor growth and enhances gemcitabine resistance in RCC by regulating hsa-miR-335/CCNB1, and silenced hsa_circ_0035483 can enhance gemcitabine sensitivity in vivo. Hsa_circ_0035483 may be the target of gemcitabine resistance in the treatment of RCC. (C) 2019 Elsevier Inc. All rights reserved.