This study aimed to assess the effects of isotemporal replacement of sitting time (SIT) with standing (STA) on cardiometabolic biomarkers. In this cross-sectional study, male adolescents wore the GT3X+ activity monitor for 7 days to measure the SIT and STA. Moderate-to-vigorous physical activity (MVPA) was estimated by a youth-specific cut-off point. An isotemporal substitution approach was used to examine the effects of replacing different periods of SIT (15, 30, 60, and 120 min) with STA on cardiometabolic biomarkers [total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), non-HDL-c, low-density lipoprotein cholesterol (LDL-c), triglycerides (TG), glucose, insulin, HOMA2-β, HOMA2-S, and HOMA2-IR]. Analysis of covariance (ANCOVA) with a post-hoc Bonferroni test was used to compare the adjusted means between the four subgroups that were clustered according to SIT and STA amount. Adolescents (n = 84; age, 16.7 ± 0.9 years) wore GT3X+ for 15.2 ± 2.3 h, for 6.7 ± 0.6 days. Isotemporal substitution of SIT with STA was associated with TC, non-HDL-c, LDL-c, and TG. ANCOVA results showed a statistically significant difference for TC, non-HDL-c, and LDL-c. These findings showed that for male adolescents, sitting less and standing more may be an effective alternative to reduce cardiometabolic biomarker levels related to lipid metabolism, regardless of MVPA.