Tujuan dari penelitian ini adalah untuk menentukan pengaruh penambahan berbagai konsentrasi tepung labu kuning dalam pembuatan roti tawar terhadap kadar air, kadar abu, kadar beta karoten dan kadar serat dan untuk menentukan konsentrasi tepung labu kuning yang paling disukai oleh panelis berdasar pada uji organoleptik. Data kadar air, kadar abu, kadar beta karoten dan kadar serat roti tawar dianalisa secara statistik dengan Rancangan Acak Kelompok (RAK) dengan 6 perlakuan dan 4 ulangan. Sebagai perlakuan adalah konsentrasi tepung labu kuning, yaitu : 0%, 2,5%, 5%, 7,5%, 10%, dan 12,5% sedang sebagai ulangan adalah waktu analisis. Hasil penelitian menunjukkan bahwa penambahan berbagai konsentrasi tepung labu kuning dapat meningkatkan kadar ari, kadar abu, kadar beta karoten, dan kadar serat roti tawar yang dihasilkan. Hasil uji organoleptik dengan 30 panelis menunjukkan bahwa penambahan tepung labu kuning yang paling disukai adalah pada konsentrasi 5% dan 7,5%, dengan kadar air 32,22 % dan 33,52 %, kadar abu 0,92 % dan 1,04 %, kadar beta karoten 5,27 mg/100g dan 6,82   mg/100g, serta kadar abu 3,33 % dan 4,62 %.The objectives of this study were to determine the effect of various pumpkin flour concentration addition in bread as revealed by water, ash, beta carotene, and fiber content, to determine the most preferred concentration of pumpkin flour applied in bread by panelists based on the organoleptic test. The water, ash, beta carotene, and fiber contents of bread were analyzed by Randomized Completely Block Design (RCBD), with 6 treatments and 4 replications. As the treatments were various concentration of pumpkin flour addition which are : 0%, 2,5%, 5%, 7,5%, 10%, and 12,5% respectively, and as blocks were time of analysis. The results showed that the addition of pumpkin flour with various concentration can increase the water, ash, beta carotene, and fiber content. The results of organoleptic test using 30 panelists showed that the most preferred concentration of pumpkin flour applied in bread 5% and 7,5% with the range of water content 32,22 % and 33,52 %, ash content 0,92 % and 1,04 %, beta carotene content 5,27 mg/100g and 6,82   mg/100g, and fiber content 3,33 % and 4,62 %.