Stimulation of myocardial β₁-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic b1-AR knockdown in three-dimensional engineered heart tissue (EHT). For β₁-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the β₁-AR (shB1) and a scrambled control (shCTR) were subcloned into a recombinant adeno-associated virus (AAV)-short hairpin RNA (shRNA) expression system. Transduction efficiency was ∼100%, and radioligand binding revealed 70% lower β₁-AR density in AAV6-shB1-transduced EHTs. Force measurements, performed over the culture period of 14 days, showed paradoxically higher force generation in AAV6-shB1 compared with shCTR under basal (0.19 ± 0.01 versus 0.13 ± 0.01 mN) and after β-AR-stimulated conditions with isoprenaline (Δfractional shortening: 72 ± 5% versus 34 ± 4%). Large scale gene expression analysis revealed that AAV6-shCTR compared with nontransduced EHTs showed only few differentially regulated genes (<20), whereas AAV6-shB1 induced marked changes in gene expression (>250 genes), indicating that β₁-AR knockdown itself determines the outcome. None of the regulated genes pointed to obvious offtarget effects to explain higher force generation. Moreover, compensational regulation of β₂-AR signaling or changes in prominent β₁-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient β₁- AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific β₁-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac β₁-AR, and 3) may open up new avenues for β-AR loss-of-function research in vivo.