Orthodontic tooth movement (OTM) is a complex biological process triggered by orthodontic forces (OF). This study aims to study the influence of sex and age on the gene expression of the dental pulp (DP) and periodontal ligament (PDL) of human premolars subjected to 7 and 28 days of OF in vivo. Linear mixed and negative-binomial models were used on previously published RNA sequencing (RNA-seq) datasets of DP and PDL tissue subjected to OF for 7 days and 28 days to verify if the effect of OF depends on sex and age. Differentially expressed genes (DEGs) were identified using false discovery rate and functional analysis was performed. The datasets consisted of 69 DP and 63 PDL samples from 46 and 41 patients respectively, with similar sex and age distribution. RNA-seq showed that sex did not influence the DP’s gene expression profile, since only one DEG related to immune response was detected after 28-days of OF. In contrast, sex significantly affected PDL: 505 DEGs were found after 7 days of OF, related to bone homeostasis, osteoclastic activity and immune response. Age impacted both tissues; in DP, 18 DEGs related to Ca2⁺ regulation and DNA damage repair were found at 7 days, and 10 DEGs associated with repair and adaptive capacities emerged at 28 days. In PDL, 181 genes related to bone regeneration were identified at 28 days, with no DEGs noted at 7 days. Our study demonstrates that under OF, DP’s reaction is not sex-based, whereas PDL’s is, particularly in the early phase of OTM, with women showing a more pronounced osteoclastic response. Age-related effects in DP tissue primarily influence Ca2+ homeostasis and DNA damage repair in early phases, and tissue repair and adaptive responses later. In contrast, age impacts PDL tissue mainly in the later stages of OTM, affecting its regenerative capacity.