The aim of this study was to evaluate the effect of intermittent parenteral copper supplementation (IPC) on serum copper status and biochemical and hematological measures of copper toxicity and deficiency in premature infants with parenteral nutrition (PN)-associated cholestasis (PNAC). We performed a prospective nested observational study in premature infants with PNAC who received IPC after the development of PNAC. Infants with chromosomal disorders, TORCH (toxoplasmosis, parvovirus, syphilis, rubella, cytomegalovirus, herpes, human immunodeficiency virus) infection, metabolic disorder, and/or surgical abnormality of the hepatobiliary system were excluded. Serum copper concentrations were measured once every 2-4 weeks while receiving PN; 24 premature infants were studied. The mean gestational age (GA) of infants was 28.6 ± 4.7 weeks. On regression analysis, there was no significant association between IPC and serum copper concentration (coefficient 2.72, 95% CI: -27 to 32; P = .84) after controlling for GA, gender, and baseline copper intake before PNAC. There was no significant association of IPC with alanine and aspartate transaminases levels (hepatotoxicity) and platelet count, hematocrit, white blood cell count, and neutrophil count (measures of copper deficiency) after controlling for confounders. GA and postmenstrual age were independently and positively associated with serum copper concentration after controlling for confounders on regression analyses. Thus, IPC in premature infants with PNAC does not influence copper status and is not associated with biochemical and hematological measures of copper deficiency and/or toxicity. Serum copper concentration in premature infants with PNAC receiving IPC is determined by the degree of prematurity and postmenstrual age.