Zinc ion (Zn²⁺) is an essential micronutrient and a potent antioxidant. However, Zn²⁺ is often limited in the environment. Upon Zn²⁺ limitation, Mycolicibacterium (basonym: Mycobacterium) smegmatis (Msm) undergoes a morphogenesis, which relies on alternative ribosomal proteins (AltRPs); i.e., Zn²⁺-independent paralogues of Zn²⁺-dependent ribosomal proteins. However, the underlying physiological changes triggered by Zn²⁺ limitation and how AltRPs contribute to these changes were not known. In this study, we expand the knowledge of mechanisms utilized by Msm to endure Zn²⁺ limitation, by comparing the transcriptomes and proteomes of Zn²⁺-limited and Zn²⁺-replete Msm. We further compare, corroborate and contrast our results to those reported for the pathogenic mycobacterium, M. tuberculosis, which highlighted conservation of the upregulated oxidative stress response when Zn²⁺ is limited in both mycobacteria. By comparing the multi-omics analysis of a knockout mutant lacking AltRPs (ΔaltRP) to the Msm wild type strain, we specify the involvement of AltRPs in the response to Zn²⁺ limitation. Our results show that AltRP expression in Msm does not affect the conserved oxidative stress response during Zn²⁺ limitation observed in mycobacteria, but AltRPs do significantly impact expression patterns of numerous genes that may be involved in morphogenesis or other adaptive responses. We conclude that AltRPs are not only important as functional replacements for their Zn²⁺-dependent paralogues; they are also involved in the transcriptomic response to the Zn²⁺-limited environment.