Objective To investigate the effect and mechnism of betaine (BET) in reversing chemotherapy resistance in prostate cancer (PCa) by inhibiting ATP-binding cassette subfamily B member 1 (ABCB1). Methods The PCa chemotherapy-sensitive C4-2B cells were cultured, and the TaxR cells resistant to docetaxel (DTX) were established by gradient increase the concentration of DTX. The drug resistance of C4-2B and TaxR cells against DTX was assessed using CCK-8 and the colony formation experiment. Western blotting and qRT-PCR were used to detect ABCB1 expression. The TaxR cells were divided into: (1) Control group, negative control group (NC), siABCB1-1 group (transfected with siABCB1-1), and siABCB1-2 group (transfected with siABCB1-2). Western blotting was used to detect the effect of small interfering RNA on silencing ABCB1, and CCK-8 was used to detect the differences in DTX resistance between each group. (2) Different concentrations of BET (0, 100, 200, 400, 600, 800 mmol/L) groups. These groups were subjected to CCK-8 to detect cell viability, and Western blotting was used to detect the protein expression of ABCB1. (3) Control group, DTX group (20 nmol/L DTX), BET group (200 mmol/L BET), and DTX+BET group (20 nmol/L DTX+200 mmol/L BET), flow cytometry was used to detect apoptosis rate and cell cycle, and Western blotting to detect the protein expression of apoptosis-related proteins (Bcl2, BAX, c-caspase-3). (4) Control group, BET group (200 mmol/L BET), wortmannin (WM) group (100 μmol/L WM), and BET+WM group (200 mmol/L BET+100 μmol/L WM). Western blotting was used to detect the protein expression of PI3K, Akt, and ABCB1. (5) Control group, BET group (200 mmol/L BET), and BAY group (10 μmol/L BAY), BAY+BET group (200 mmol/L BET+10 μmol/L BAY). Western blotting was used to detect the protein expression of NF-κB p65, p-ikBα and ABCB1. Network pharmacology combined with transcriptome sequencing was used to predict the possible pathways for BET to reverse chemotherapy resistance. Results Compared with C4-2B cells, TaxR cells showed significantly increased resistance to DTX (P<0.01), and high expression of ABCB1 (P<0.01). After silencing ABCB1 with siRNA, TaxR cells' resistance to DTX was significantly inhibited (P<0.01). The inhibition rate of TaxR cells treated with 200 mmol/L BET was less than 20%, and it significantly decreased the expression of ABCB1 protein in TaxR cells (P<0.05). Compared with control group, the combination of 200 mmol/L BET and 20 nmol/L DTX resulted in higher apoptosis rate and higher S stage cell ratio, lower expression of Bcl-2 protein and higher expression of BAX and c-caspase-3 proteins than the two drugs used alone (P<0.05). Compared with control group, the combination of 200 mmol/L BET and 100 μmol/L WM significantly down-regulated the protein expression of PI3K, Akt and ABCB1 (P<0.01). The combination of 200 mmol/L BET and 10 μmol/L BAY significantly down-regulated the protein expression of NF-κB p65, p-ikBα and ABCB1 (P<0.01). Conclusion BET may reverse TaxR cells' chemotherapy resistance by down-regulating ABCB1 expression through the PI3K/Akt/NF-κB signaling pathway.