Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a critical protein for photosynthesis. With small subunits, RBCSs catalyze the fixation of CO2 onto ribulose-1,5-bisphosphate (RuBP). However, the gene characteristics of peanut RBCSs, and the relationship between their expression and peanut growth under nitrogen (N)-deficiency conditions are not yet known. This study targeted the impact of sufficient N (5 mmol/L NO3−) and deficient N (0.1 mmol/L NO3−) on peanut seedlings’ growth, physiology, and molecular aspects. Herein, eight RBCS protein encoding genes were identified in the peanut genome, and their characteristics were described. The leaf areas of Yu Hang Hua 1 (YH1) and An Hua 3 (AH3) were reduced significantly after 10 days while “Pu Ke Hua 18” (PK18) and “Luo Hua 4087” (LH4087) had higher N-deficiency adaptations in the initial N-deficiency stage. The Rubisco carboxylation activities of YH1 and PK18 were significantly increased after 5 days of N-deficiency stress while they decreased under 15 days of N-deficiency conditions. Furthermore, the qRT-PCR results showed that the expressions of AhRBCS 1/5 and AhRBCS2/6 had negative relationships with the morpho-physiological traits of peanut under low-N stress. This study provides the biological information of peanut RBCSs and lays a theoretical foundation for N utilization in crop plants.