Abstract: In order to improve the effect of hydroxychloroquine (HCQ) on the selectivity in anti-tumour therapy, the α-linolenic acid conjugated hydroxychloroquine (AHQ) was designed, which was aiming to enhance anti-tumor effect. Basing on the in vitro data of AHQ, we chose colon cancer as the model disease. Herein, we assumed the combined therapy with 5-fluorouracil(5-Fu), which was the corner-stone on the treatment of colon cancer. After the successful synthesis, aqueous solubility, stability of ester bond and in vivo acute toxicity analysis of AHQ, the in vivo bio-distribution of AHQ in the tumour bearing mice model was constructed to verify the increased selectivity in the tumour tissue. The AHQ was demonstrated better tumour selectivity in the HT-29 bearing mice model than HCQ. The synergism effect and best proportion between AHQ and 5-Fu against HT-29 and HCT116 cell lines were 1:1 and 1:8, respectively. The combined group could enhance HT-29 cell apoptosis significantly via arresting the cell cycle in the G0/G1 phase comparing with both AHQ and 5-Fu, while it improved the HCT116 cell apoptosis significantly via arresting the cell cycle in the S phase compared to AHQ. Basing on the prominent consequence in the HT-29 cell line, we naturally chose HT-29 xenograft mouse model to continue the study. The combination group showed excellent effect on suppressing tumour growth by up-regulating the expression of LC3-Ⅱ and P62, and inhibiting the pathway of Akt/mTOR in autophagy process.