Prostate cancer is closely associated with constitutive transactivation of the androgen receptor (AR) signaling pathway. After treatment with androgen‑deprivation therapy (ADT), the majority of patients develop castration‑resistant prostate cancer within months or years. In order to investigate potential novel therapeutic targets in addition to ADT, the present study examined the regulatory mechanisms of the AR signaling pathway. In the present study, LNCaP cells were metabolically‑labeled with Alk‑C16, a palmitate probe. In addition, cells were treated with R1881, an androgen, or DMSO. Subsequently, click‑chemistry‑based palmitoylome profiling was performed in LNCaP cells and palmitoylated proteins were compared between cells treated with androgen and untreated cells. Androgen treatment was revealed to significantly increase the palmitoylation level of α‑tubulin. In addition, the palmitoylation level of Ras‑related protein Rab‑7a (Rab7a) was enhanced by androgen treatment. Palmitoylation of α‑tubulin and Rab7a were essential for cell proliferation. Notably, in the supernatant of LNCaP cells, the palmitoylation level of α‑tubulin was also increased following androgen treatment. Palmitoylation of α‑tubulin may provide a new potential target for the treatment of prostate cancer. In addition, the high level of α‑tubulin palmitoylation in the supernatant may represent a biomarker for early‑stage prostate cancer.