<p>Grass carp (<i>Ctenopharyngodon idellus</i>) is an economically important fish, produced at the highest level among all freshwater aquaculture fish in the world, yet the significant number of intermuscular bones (IBs) present in the fillets severely limits its consumption and processing options. To address this limitation, we first investigated the developmental characteristics of IBs in grass carp. Our results revealed that the key period for IB ossification from myosepta occurred between 15 and 40 days post-hatching (dph). Through gene editing of <i>runx2b</i>, the key gene for IB development, we successfully constructed an F₀ population with <i>runx2b</i> mutations, and the F₁ generation was created through breeding of female and male founders. Subsequently, individuals that completely lacked IBs were screened out from F₁ generation, and then F₂ generations completely lacked IBs were bred. Micro-CT showed that the loss of IBs caused by the <i>runx2b</i> mutation did not affect the formation or mineral density of other main skeletal elements (<i>P</i>&gt;0.05) or the proportion of fat or muscle volume (<i>P</i>&gt;0.05). Nutritional characteristic profiling further revealed no significant differences between the <i>runx2b</i> mutant grass carp without IBs and the wild-type grass carp with IBs in terms of moisture, crude protein, crude fat, amino acids, fatty acids, total sugars, collagen, or other nutrients (<i>P</i>&gt;0.05). An integrated analysis of transcriptomic, proteomic, and metabolomic data provided important information regarding the genetic regulation of nutritional indicators of muscle in grass carp without IBs. Changes in the expression of genes related to Ca²⁺ signaling and movement indicated that the adaptation of grass carp without IBs leads to increased muscle contraction. These data provide an essential reference for the quality assessment of fish lacking IBs and indicate that this new strain of grass carp has great potential for improving its seeds and processing industry.</p>