Updated project metadata. Galiellalactone (GL) is a fungal metabolite that presents antitumor and anti-inflammatory activities in vitro and in vivo. Previous studies have shown that GL targets NF-KB and STAT3 pathways and induces G2/M cell cycle arrest in androgen-insensitive prostate cancer cells. In this study, we show that GL-induced cell cycle arrest is independent of the NF-KB and STAT3 pathways in DU145 and PC-3 cells, and also that GL did not affect cell cycling in androgen-sensitive prostate cancer cell such as LNCaP and 22Rv1 cells. In addition, we showed confluence resistance to GL in DU145 cells. Using a SWATH proteomic approach we identified the down-regulation of Nucleolar and spindle associated protein 1 (NUSAP1) under DU145 confluence and in LNCaP cells. Also, the inhibition of NUSAP1 by siRNAs induced resistance to GL in DU145 cells, suggesting that NUSAP1 may be a target for GL and could be useful as biomarker for responsiveness of the antitumor activity of GL.