Lucerne is regarded as the best legume crop for forage to be cultivated in South Africa. It is commonly used to produce good quality hay. It also plays an important role in soil conservation, regeneration, and crop rotation systems as it supplies substantial amounts of nitrogen to succeeding crops through symbiotic N2 fixation, which makes it the preferable choice for intensive forage production systems. Fertilizer in liquid inoculant formulations has demonstrated to contribute growth and yield increase for leguminous crops. Therefore, the aim of this paper was to determine the effects of Sinorhizobium meliloti liquid formulation inoculation on the growth, yield, and nitrogen content in lucerne. The strain RF14 (Sinorhizobium meliloti) was collected from the Agricultural Research Council at Roodeplaat (Plant Health and Protection located (East), Pretoria (South Africa). The liquid inoculant contained 6.5 × 109 viable cells mL−1. According to the Kooen–Gieger climatic classification, the experiments were conducted on two different climatic zones. The first site was in Bronkhorspruit (Blesbokfontein farm) in the Gauteng province and the second was in Hartbeesfontein (Rietfontein Farm) in the Northwest province. The results showed that lucerne inoculation with liquid inoculant formulation of Sinorhizobium meliloti significantly increased nodule number, size, growth, and yield in both bioclimatic zones. The significantly increased were compared to the negative control. The Sinorhizobium meliloti inoculant increased nitrogen accumulation in all inoculated treatments compared to the control. The finding of this research provides important information on the impact of rhizobium microbial inoculant application in the improvement of soil fertility through nodule formation. In addition, seed vigor improvement was translated in overall growth and yield increase in lucerne plants.