Background Preterm infants received long-term parenteral nutrition (PN) due to gastrointestinal immaturity. Mixed lipid emulsions composed of soybean oil, medium chain triglycerides (MCTs), olive oil, and fish oil, which have a relatively low ω-6: ω-3 ratio, may decrease the incidence of PN-associated cholestasis by reducing oxidative stress and providing an anti-inflammatory effect.Methods The retrospective cohort study enrolled a total 399 very low birth weight (VLBW) premature infants between January 2009 and November 2017 at a single neonatal intensive care unit. Preterm infants received total parenteral nutrition with either mixed lipid emulsion (SMOFlipid, n = 195) or soybean oil-based lipid emulsion (Lipovenoes, n = 204) for at least seven days. We compared the outcomes of PN-associated cholestasis, co-morbidities and mortality.Results The incidence of PN-associated cholestasis was significantly lower in the SMOFlipid group than the Lipovenoes group (10.3% vs 20.1%, P = 0.006). The related clinical laboratory findings, including levels of γ- GT (P = 0.019), triglyceride (P < 0.001), and cholesterol (P = 0.023), were significantly lower in the SMOFlipid group. The duration to full feeding days shortened in the SMOFlipid group compared with the Lipovenoes group (25 ± 10.33 vs 33 ± 16.22, P < 0.001). Relevant complications, such as severe retinopathy of prematurity (ROP, 3.6% vs 14.3%, P < 0.001) and bronchopulmonary dysplasia (BPD, 36.9% vs 46.7%, P = 0.046) were also reduced in the SMOFlipid group versus the Lipovenoes group, but there was no significant effect on severe intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), or mortality in both groups.Conclusions In very premature infants, PN with fish oil-based lipid emulsions is associated with a lower incidence of PN-associated cholestasis compared with soybean oil-based lipid emulsions.