Onivyde is the first irinotecan (IRI) nanoliposome that could improve pharmacokinetics and tumor biodistribution of irinotecan. Although FDA approves Onivyde for the treatment of pancreatic cancer patients who are not effective for GEM, the gastrointestinal toxicity caused by Onivyde is still a problem to be solved in clinical application. Berberine (BER), an isoquinolone alkaloid extracted from several different plants, has been reported to exhibit beneficial effect in alleviating intestinal mucositis and generating synergistic anticancer effect in combination with cytotoxic drugs. However, its therapeutic effect is affected by the different pharmacokinetic behavior of two drugs. Therefore, we utilized triethylamine-sucrose octasulfate gradient to construct nanoliposomes for co-delivery of irinotecan and berberine, termed as lipBI. This co-delivery nanoliposomes remained the synergistic ratio in the body and improved tumor distribution of IRI and BER. The lipBI significantly inhibited tumor growth in the BXPC-3 pancreatic cancer model compared with Onivyde (p < 0.05) and reduced the gastrointestinal toxicity in mice caused by IRI. Overall, IRI/BER co-loaded liposomes possessed great potential in the treatment of pancreatic cancer.