Introduction The effect of isotretinoin on inflammatory markers has been reported with conflicting results. No studies have been reported on the relationship between isotretinoin and lymphocyte/high-density lipoprotein cholesterol [HDL-C] ratio (LHR), neutrophil/HDL-C ratio (NHR), or platelet/HDL-C ratio (PHR) in acne patients. Objectives We aimed to investigate how isotretinoin affects the inflammatory markers, including LHR, NHR, and PHR, in acne vulgaris patients. Methods A total of 361 patients with moderate-to-severe acne vulgaris who received systemic isotretinoin for at least six months were included. Complete blood count and biochemical analyses, including monocyte/HDL-C ratio (MHR), NHR, LHR, PHR, neutrophil/lymphocyte ratio (NLR), monocyte/lymphocyte ratio (MLR), platelet/lymphocyte ratio (PLR), the aggregate index of systemic inflammation (AISI), systemic immune-inflammation index (SII), and systemic inflammation response index (SIRI), obtained at the treatment onset and the end of the first, third, and sixth months of treatment were investigated. Results There was no significant difference in MHR and NHR levels between repeated measures. A significant increasing trend was seen in LHR and PHR (P = 0.001 and P = 0.011, respectively). HDL-C levels gradually and significantly declined during the six months (P < 0.001). Serum NLR, derived NLR, SII, SIRI, MLR, and AISI showed a significant decrease in line with clinical improvement in acne during the six months of therapy (P < 0.05). Conclusions Declining levels of NLR, MLR, SII, SIRI, and AISI may indicate the anti-inflammatory effects of isotretinoin on the pilosebaceous unit, whereas increasing levels of LHR and PHR may show systemic inflammatory activity of isotretinoin.