This study investigated the biochemical and histopathological alterations along with the immunoexpression pattern of heat shock protein 27 (Hsp27) within 6 h postmortem (PM) in skeletal muscle of boldenone (BOL)- treated rats. Forty-eight male rats were divided into two groups; a control group received sesame oil (0.25 mL/kg bwt), and BOL group received 5 mg/kg bwt BOL. Both treatments were intramuscularly injected once a week for eight weeks. Rats were euthanized by cervical dislocation, and the skeletal muscle specimens were collected at zero-time, 2, 4, and 6 h PM for biochemical and histopathological evaluations. The results revealed that BOL treatment significantly increased pH, MDA, ATP, ADP, glycogen, and hydroxyproline values. Still, it decreased the GPX, GST, and lactic acid levels, and Hsp27 immunoexpression compared to the control group. With increasing postmortem interval (PMI), whether control or BOL-treated, a significant reduction in pH value, markers of muscular antioxidant status, ATP, ADP, glycogen, hydroxyproline levels, as well as Hsp27 immunoexpression but a significant increase in lipid peroxidation and lactic acid content were recorded. Of note, the interaction between BOL treatment and PMI had a significant effect on ATP, ADP, lactic acid, hydroxyproline, GST, MDA, and TAC levels. Conclusively, these findings signify BOL exposure’s modifying effect on the energy content, oxidative status, and histological architecture of skeletal muscles in the early PMI that reflected in delaying the onset of rigor mortis. For forensic practitioners, these findings should be highly considered at estimating PMI in athletic, AAS-treated patients, and fattening animals.