Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca 2+ sensitivity. Mouse models exhibit increased Ca 2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca 2+ buffering and altered Ca 2+ handling contribute to HCM pathogenesis via activation of Ca 2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca 2+ handling and Ca 2+-dependent signaling in a model system possessing Ca 2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the K d of Ca 2+ binding, resulting in higher Ca 2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca 2+] and slowed Ca 2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca 2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca 2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca 2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal–regulated kinase pathways. Altered myofilament Ca 2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca 2+ sensitivity provides an attractive therapeutic approach in HCM.