Uniform actin filament length is required for synchronized contraction of skeletal muscle. In myopathies linked to mutations in tropomyosin (Tpm) genes, irregular thin filaments are a common feature, which may result from defects in length maintenance mechanisms. The current work investigated the effects of the myopathy-causing p.R91C variant in Tpm3.12, a tropomyosin isoform expressed in slow-twitch muscle fibers, on the regulation of actin severing and depolymerization by cofilin-2. The affinity of cofilin-2 for F-actin was not significantly changed by either Tpm3.12 or Tpm3.12-R91C, though it increased two-fold in the presence of troponin (without Ca). Saturation of the filament with cofilin-2 removed both Tpm variants from the filament, although Tpm3.12-R91C was more resistant. In the presence of troponin (±Ca), Tpm remained on the filament, even at high cofilin-2 concentrations. Both Tpm3.12 variants inhibited filament severing and depolymerization by cofilin-2. However, the inhibition was more efficient in the presence of Tpm3.12-R91C, indicating that the pathogenic variant impaired cofilin-2-dependent actin filament turnover. Troponin (±Ca) further inhibited but did not completely stop cofilin-2-dependent actin severing and depolymerization.