Introduction: Insertion of Peripheral intravenous catheter (PIVC) is a common invasive procedure performed on patients that may contribute to several complications. Nurses are required to have adequate knowledge and practice to ensure the successful of the procedure. The objective of this study was to determine the knowledge and practice of nurses towards PIVC insertion procedures. Methods: A descriptive cross-sectional study design was conducted at Universiti Kebangsaan Malaysia Medical Centre. A total of seventy- seven nurses working in the emergency department, obstetrics & gynecology patient admission centre, daycare oncology were participated in this study. The data was gathered using a modified questionnaire and standard PIVC insertion guidelines. Results: 21 (27.3%) nurses had high knowledge and 56 (72.7%) had low knowledge of PIVC insertion (M=7.77, SD=1.87). All 77 (100%) nurses performed insertion of PIVC according to the standard of practice (M=23.16, SD=1.51). A positive correlation between age and knowledge (rho=0.123) but a negative correlation towards practice (rho=-0.413). A positive correlation between working experience and knowledge (rho=0.094) but a negative correlation towards practice (rho=-0.047). There was a positive correlation between nurses’ knowledge and practice (rho=0.038). Conclusion: Nurses with good knowledge of PIVC insertion performed better as they were aware of the negative consequences of the procedure if conducted incorrectly. Knowledgeable and competent nurses are essential to ensure the quality of nursing care and safe practice towards patients. Future studies with larger samples of nurses involved in more hospitals in Malaysia are recommended.